Relaxin produces a sustained decrease in total peripheral resistance, but the effects of relaxin on skeletal muscle arterioles, an important contributor to systemic resistance, are unknown. Using the intact, blood-perfused hamster cremaster muscle preparation in situ, we tested the effects of relaxin on skeletal muscle arteriolar microvasculature by applying 10(-10) M relaxin to second-, third- and fourth-order arterioles and capillaries. The mechanisms responsible for relaxin-induced dilations were explored by applying 10(-10) M relaxin to second-order arterioles in the presence of 10(-5) M N(G)-nitro-l-arginine methyl ester (l-NAME, nitric oxide (NO) synthase inhibitor), 10(-5) M glibenclamide (GLIB, ATP-dependent potassium (K(+)) channel inhibitor), 10(-3) M tetraethylammonium (TEA) or 10(-7) M iberiotoxin (IBTX, calcium-associated K(+) channel inhibitor). Relaxin caused second- (peak change in diameter: 8.3 ± 1.7 μm) and third (4.5 ± 1.1 μm)-order arterioles to vasodilate transiently while fourth-order arterioles did not (0.01 ± 0.04 μm). Relaxin-induced vasodilations were significantly inhibited by l-NAME, GLIB, TEA and IBTX. Relaxin stimulated capillaries to induce a vasodilation in upstream fourth-order arterioles (2.1 ± 0.3 μm), indicating that relaxin can induce conducted responses vasodilation that travels through blood vessel walls via gap junctions. We confirmed gap junction involvement by showing that gap junction uncouplers (18-β-glycyrrhetinic acid (40 × 10(-6) M) or 0.07% halothane) inhibited upstream vasodilations to localised relaxin stimulation of second-order arterioles. Therefore, relaxin produces transient NO- and K(+) channel-dependent vasodilations in skeletal muscle arterioles and stimulates capillaries to initiate conducted responses. The transient nature of the arteriolar dilation brings into question the role of skeletal muscle vascular beds in generating the sustained systemic haemodynamic effects induced by relaxin.