Previous studies from this laboratory suggest that during maturation, rapid microvascular growth is accompanied by changes in the mechanisms responsible for regulation of tissue blood flow. To further define these changes, we studied isolated gracilis muscle arterioles from weanling (~25 days) and juvenile (~44 days) Sprague Dawley rats to test the hypothesis that endothelial mechanisms for the control of arteriolar tone are altered with growth. Responses to the endothelium-dependent dilator acetylcholine (ACh) were greater in weanling arterioles (WA) than in juvenile arterioles (JA), whereas there were no consistent differences between age groups in arteriolar responses to other endothelium-dependent agonists (A23187, VEGF and simvastatin). Inhibition of nitric oxide synthase (NOS) with L-NAME attenuated ACh-induced dilation in JA, but not in WA. In JA, combined inhibition of NOS and cyclooxygenase (with indomethacin) reduced the dilator responses to ACh and simvastatin by ~90% and ~70%, respectively, but had no effect in WA. Cytochrome P450 epoxygenase inhibition (with PPOH) had no effect on responses to ACh or simvastatin in either age group. Inhibition of Ca²⁺-activated or ATP-dependent potassium channels (with TEA or glibenclamide, respectively) reduced these arteriolar responses in JA but not WA. These findings suggest that in fully grown microvascular networks, endothelium-dependent arteriolar dilation is mediated by the combined release of endothelial nitric oxide and vasodilator prostanoids, and in part through activation of K_Ca and K_ATP channels. However, during earlier microvascular growth, this dilation is mediated by other factors yet to be identified. This may have significant implications for the regulation of tissue perfusion during microvascular development.