The distribution, ontogeny and role of P2x1 receptors were examined in the smooth muscle of the mouse intestine, bladder, and male and female reproductive tracts using P2x1 receptor subtype selective antibodies and contraction studies. P2x1 receptor immunoreactivity showed a heterogeneous distribution in smooth muscle with high levels expressed in adult vas deferens, bladder, arteries and male reproductive organs. In contrast, P2x1, receptors were below the level of detection in the smooth muscle of the ileum and female reproductive tract. P2x1 receptor immunoreactivity was detected at adult levels from birth in the bladder. However, in the vas deferens, immunoreactivity was only detected from 10 days after birth and reached adult levels by approximately 1 month old. A similar pattern of expression was seen in the vesicular seminalis, epididymis, gland of the vas deferens and coagulating gland. Sensitivity to the P2x1 receptor agonist alpha,beta-methylene ATP (alpha,beta-meATP) and P2x1 receptor-deficient mice were used in functional studies to determine the role of P2x1 receptors in the control of smooth muscle. alpha,beta-meATP (100 microM) failed to evoke contractions of the epididymis, or seminal vesicle and P2x1 receptors did not contribute to the control of uterine smooth muscle. In the ileum, alpha,beta-meATP (100 microM) evoked a transient relaxation followed by a contraction. These responses were abolished by the P2 receptor antagonist iso-pyridoxalphosphate-6-azophenyl-2'-5'-disulphonate (iso-PPADS) (30 microM). Relaxant responses were abolished by the adenosine A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (1 microM). Contractile responses were reduced by > 80% in the ileum from P2x1 receptor-deficient mice. alpha,beta-meATP-evoked contractions were reduced by approximately 35% by TTX (1 microM) and were unaffected by atropine (10 microM). These studies indicate that P2x1 receptors are not expressed throughout all smooth muscles and that their expression is developmentally regulated. In addition, they provide evidence to suggest that P2x1 receptors are present on pre-synaptic nerve terminals in the enteric nervous system.