Both in vitro and in vivo studies have revealed that removal of vascular endothelial cells abolishes the vasodilation to acetylcholine (Ach) but not sodium nitroprusside (SNP). Differential properties of endothelial cells in the series-arranged arterioles to vasodilator responses have not been studied. In this study, the cheek pouch microcirculation from the golden syrian hamster anesthetized with sodium pentobarbital (6 mg/100 g body wt, ip) was prepared for intravital microscopy. Measurements of lumen diameters of small series-arranged arterioles (2nd- and 4th-order) were made before, during, and after topical microapplication of different doses of either Ach or SNP. After control measurements, a light-dye (L-D) technique utilizing sodium fluorescein (FITC-dextran(150K), 50 mg/100 g body wt, iv) and illuminating a discrete area of the arteriole with 490-nm-wavelength light for 3 (4th) or 10 (2nd) min was used to impair endothelial cell function without damaging vascular smooth muscle cells. Responses to vasoactive substances for both 4th-order (10-20 microns) and second-order (30-50 microns) arterioles were retested. Vasodilatory responses to 10(-7) M Ach and SNP also were tested with and without the presence of NG-monomethyl L-arginine (L-NMMA), an inhibitor of EDRF/NO formation. In the control state, Ach and SNP produced a focal, dose-dependent increase in diameter in all arterioles tested. Endothelial impairment by L-D treatment significantly suppressed the vasodilator response to Ach in 4th- but not 2nd-order arterioles, whereas the SNP response was not significantly affected. Consistent with these observations, L-NMMA treatment significantly attenuated Ach-induced vasodilation in 4th-order arterioles, but it had no effect on 2nd-order arterioles. These studies document further the role of the endothelium in local modulation of arteriolar diameter in response to acetylcholine and demonstrate a differential effect for this response in series-arranged microvessels. Thus, there may be a heterogeneous distribution of endothelial cell functions for modulating vasodilator activity in microvessels.