Microvilli isolated from 13762 mammary ascites tumor cells contain a major calcium‐sensitive protein (AMV‐p35) that can be isolated with microvillar microfilament cores prepared by Triton X‐100 extraction in the presence but not absence of calcium. AMV‐p35 can be readily purified from ethylene glycol bis(β‐aminoethyl ether)‐N,N,N‘,N‘‐tetraacetic acid extracts of the microfilament cores by chromatography on an anion exchange column, to which it does not bind. Immunoblot analysis indicates that AMV‐p35 is related to calpactin I, the pp60srctyrosine kinase substrate. In the presence of calcium, AMV‐p35 binds approximately 4 mol of chlorpromazine per mole of protein in a binding process showing apparent positive cooperativity, similar to calmodulin; however, in contrast to calmodulin, AMV‐p35 also binds phenothiazine in the absence of calcium.—Carraway, K. L., III; Liu, Y.; Puett, D; Carraway, K. L.; CarothersCarraway, C. A. Phenothiazine binding by a homolog of calpactin, the pp60srctyrosine kinase substrate.FASEB J.1: 46‐50; 1987.