Actin in cultured bovine retinal capillary pericytes was identified and partially characterized biochemically. The filamentous actin was localized in bovine retinal capillary pericytes using a fluorescent mushroom toxin (nitrobenzoxadiazole-phallacidin) specific for actin. One-dimensional SDS-polyacrylamide-gel electrophoresis of urea-extracted proteins from bovine retinal capillary pericytes revealed a 46,000 MW protein band corresponding to an actin standard, which comprised 7.3% of the total urea-soluble proteins. Actin-activated skeletal muscle myosin Mg2+-ATPase assay, using [gamma-32P]-ATP as substrate, demonstrated functional actin in bovine retinal capillary pericyte extracts after DEAE-cellulose anion-exchange chromatography. The actin-containing protein fractions were eluted at ionic strengths between 0.25 and 0.35 M KCl. The presence of functional actin in pericytes indicated the ability to generate contractile force. This contraction-generating ability may allow pericytes to regulate microvessel caliber and to maintain the integrity of the capillary wall. A lack of this function when pericytes are preferentially lost in diabetic retinal microangiopathy could destabilize the microvessel wall and predispose the capillary to further pathologic changes.