We examined whether contraction-induced muscle microvascular recruitment would expand the surface area for insulin and nutrient exchange and thereby contribute to insulin-mediated glucose disposal. We measured in vivo rat hindlimb microvascular blood volume (MBV) using contrast ultrasound and femoral blood flow (FBF) using Doppler ultrasound in response to a stimulation frequency range. Ten minutes of 0.1-Hz isometric contraction more than doubled MBV (P < 0.05; n = 6) without affecting FBF (n = 7), whereas frequencies >0.5 Hz increased both. Specific inhibition of nitric oxide (NO) synthase with N(omega)-l-nitro-arginine-methyl ester (n = 5) significantly elevated mean arterial pressure by approximately 30 mmHg but had no effect on basal FBF or MBV. We next examined whether selectively elevating MBV without increasing FBF (0.1-Hz contractions) increased muscle uptake of albumin-bound Evans blue dye (EBD). Stimulation at 0.1 Hz (10 min) elicited more than twofold increases in EBD content (micrograms EBD per gram dry tissue) in stimulated versus contralateral muscle (n = 8; 52.2 +/- 3.8 vs. 20 +/- 2.5, respectively; P < 0.001). We then measured muscle uptake of EBD and (125)I-labeled insulin (dpm per gram dry tissue) with 0.1-Hz stimulation (n = 6). Uptake of EBD (19.1 +/- 3.8 vs. 9.9 +/- 1; P < 0.05) and (125)I-insulin (5,300 +/- 800 vs. 4,244 +/- 903; P < 0.05) was greater in stimulated muscle versus control. Low-frequency contraction increases muscle MBV by a NO-independent pathway and facilitates muscle uptake of albumin and insulin in the absence of blood flow increases. This microvascular response may, in part, explain enhanced insulin action in exercising skeletal muscle.