Objectives were to identify PKC isoforms in iris sphincter isolated from rabbit, cat, dog and bovine irides, to determine their sub-cellular distribution, and to investigate the effects of the phorbol ester, PDBu, on contraction and cAMP accumulation in this tissue. Using six isoform (α,β,γ,ϵ,δ,ζ)-specific polyclonal antibodies, PKCα,β,ϵ,δ, andζwere detected in the four species, whereas PKCγwas detected only in dog and bovine. PKCαandϵare the most abundant isoforms in this tissue. PKCαis mainly cytosolic in rabbit and bovine and membrane associated in cat and dog. PKCγis equally distributed in cytosol and membrane fractions of bovine, but mostly cytosolic in dog. PKCβ,δandϵare mainly membraneous and PKCζis mainly cytosolic in all species. PDBu (100 nM) induced a contractile response in rabbit-and cat-, but not in dog and bovine, sphincters, and increased cAMP accumulation in rabbit, cat, dog and bovine by 111, 130, 458 and 294%, respectively. Therefore, the lack of effect of PDBu on contraction in dog and bovine, as compared to rabbit and cat, may be due: (a) to the presence of PKCγisoform, and (b) to the stronger stimulatory effects of the phorbol ester on cAMP production in the non-contracting species. In addition to demonstrating the presence of various PKC isoforms in the iris sphincter and the activation of adenylyl cyclase by this protein kinase, we have shown that the distribution of the PKC isoforms in this tissue is species specific. Furthermore, our data suggest that there may be specific physiological functions associated with each of the PKC isoforms and that PKC is involved in the contractile response of some but not all smooth muscles.