Inositol triphosphate (IP3) and diacylglycerol (DG) are second messengers which control ionic events implicated in cell proliferation in a variety of tissues. In order to determine if these two second messengers control the proliferation of bovine retinal capillary pericytes (BRCP) or feline retinal pigment epithelial cells (FRPE) in culture, both intact BRCP or FRPE or BRCP or FRPE made permeable by saponin were used to study the effects of IP3 and DG on [3H]thymidine incorporation into DNA. [3H]Thymidine incorporation by BRCP made permeable to saponin showed specific IP3 dose-dependence; the apparent Km was 0.3 microM of IP3. Similar effects of A23187, a Ca2+ ionophore, or synthetic DG (1-oleoyl-2 acetyl-glycerol) were also observed. The combination of synthetic DG (0-,2-,4-, 8 micrograms ml-1) and 1 microM A23187 produced greater stimulation of [3H]thymidine incorporation by intact BRCP than was seen with DG or A23187 alone. In contrast to BRCP. [3H]thymidine incorporation by FRPE was not stimulated by IP3, A23187 or synthetic DG. The synergistic activation of IP3 and DG provided direct evidence to support the view that BRCP proliferation in vitro were regulated by the levels of the two second messengers.