We examined whether metabolic inhibition (5 mM NaCN + 10 mM 2-deoxyglucose) affects sarcolemmal fluidity in cultured neonatal cardiomyocytes. As a measure of sarcolemmal fluidity we determined the fluorescence steady-state anisotropy (rss, which is reciprocally related to membrane fluidity) of cardiomyocytes labeled with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene, p-toluenesulfonate. During metabolic inhibition, membrane fluidity increased progressively: after 30 min rss had fallen by 6.7 +/- 1.2% (mean +/- SE; n = 9; P < 0.05) compared to baseline values, and after 90 min by 14.5 +/- 3.5% (P < 0.05; n = 5). Beyond 90 min rss did not decrease any further. During control incubations (without metabolic inhibition), no significant changes in rss were observed. During metabolic inhibition cellular free cholesterol content declined: after 30 min free cholesterol content had decreased by 12.2 +/- 3.1% (P < 0.02; n = 4), compared to baseline values, and after 90 min by 31.1 +/- 8.3% (P < 0.02; n = 4). We conclude that metabolic inhibition induces an increase in sarcolemmal fluidity, which may be caused by a decrease in sarcolemmal free cholesterol content.