In developing technetium-99m-based radioligands for in vivo studies of cardiac adrenergic neurons, we compared the uptake characteristics of the (99m)Tc-labeled 1-(4-fluorobenzyl)-4-(2-mercapto-2-methyl-4-azapentyl)-4-(2-mercapto-2-methylpropylamino)-piperidine ((99m)Tc-FBPBAT) with those of the clinically established meta-[(123)I]iodobenzylguanidine ((123)I-MIBG) in rat vascular smooth muscle cells and neonatal cardiac myocytes. Furthermore, the cardiac and extracardiac uptake of both radiopharmaceuticals was assessed in intact rats and in rats pretreated with various alpha- and beta-adrenoceptor drugs, and adrenergic reuptake blocking agents. The uptake of (99m)Tc-FBPBAT and (123)I-MIBG into vascular smooth muscle cells and neonatal cardiac myocytes was rapid; more than 85% of the radioactivity accumulation into the cells occurring within the first 3 minutes. Radioactivity uptake after a 60-minute incubation at 37 degrees C (pH 7.4) varied from 15% to 65% of the total loaded activity per million cells. In all cases, (99m)Tc-FBPBAT showed the higher uptake, relative to (123)I-MIBG, at any given cell concentration. The cellular uptake of (99m)Tc-FBPBAT was lower at 4 degrees C and 20 degrees C than at 37 degrees C. In contrast, the (123)I-MIBG uptake was only slightly temperature dependent. Inhibition experiments confirmed that the cellular uptake of (123)I-MIBG is mediated by the uptake-I carrier, whereas alpha(1)- and beta(1)-adrenoceptors were predominantly involved in the uptake of (99m)Tc-FBPBAT into the cardiovascular tissues. Biodistribution studies in rats showed that (99m)Tc-FBPBAT accumulated in myocardium after intravenous injection. Radioactivity in rat heart amounted to 2.32% and 1.91% of the injected dose per gram at 15 and 60 minutes postinjection, compared with 3.10% and 2.21% injected dose per gram of tissue (%ID/g) in the experiment with (123)I-MIBG, respectively. Prazosin, urapidil, and metoprolol were as effective as treatment with other adrenergic drugs in lowering cardiac uptake of (99m)Tc-FBPBAT. Uptake reduction was more pronounced in myocardium than in other adrenergic-rich organs, including the lung, spleen, kidney, and adrenals, suggesting that the (99m)Tc-FBPBAT uptake in myocardium specifically reflects a high degree of alpha(1)/beta(1)-receptor binding to cardiac adrenergic neurons. In comparison, reduction of cardiac and pulmonary uptake of (123)I-MIBG was effective after pretreatment of rats with desipramine and reserpine, confirming distinct neuronal binding sites for (99m)Tc-FBPBAT and (123)I-MIBG. (99m)Tc-FBPBAT was excreted via urine and to a lower degree via feces. Urine analysis 6 hours p.i. revealed that more than 40% of the total excreted radioactivity was unmetabolized (99m)Tc-FBPBAT. In conclusion, the uptake of (99m)Tc-FBPBAT in rat myocardium specifically reflects binding to cardiac adrenergic neurons. The (99m)Tc-FBPBAT uptake appears to be predominantly mediated via the alpha(1)/beta(1)-adrenoceptor pathway. These data indicate that (99m)Tc-FBPBAT, like (123)I-MIBG, may be suitable for mapping cardiac adrenergic innervation by SPET, especially for alpha(1)/beta(1)-adrenoceptors as target in numerous heart diseases.