The Src family tyrosine kinase, Fyn, can facilitate regulation of cell proliferation and differentiation. Mice with mutations in the fyn gene have defects in the brain, immune system, and epidermal differentiation. To identify molecules that may interact with Fyn in the epidermis, we performed a yeast two-hybrid interaction screen of a murine keratinocyte library. A novel adaptor-like molecule was isolated and termed Srcasm for Src activating and signaling molecule. Murine Srcasm is a 52.7-kDa protein that contains a VHS membrane association domain and a number of tyrosine motifs suggesting that it may be a substrate for Src family kinases and serve as an adaptor protein. Northern blot analysis of murine tissues demonstrates that Srcasm expression is highest in brain and kidney. In situ hybridization analysis reveals that srcasm mRNA is expressed in regions of the epidermis and hair follicle where keratinocyte differentiation occurs. In the brain, srcasm mRNA distribution correlates with that of fyn, with both being highly expressed in the hippocampal and cerebellar Purkinje neurons. Fyn can phosphorylate Srcasm, and association of these molecules relies on cooperative binding between the SH2 and SH3 domains of Fyn and corresponding canonical binding sites in Srcasm. Srcasm is capable of interacting with Grb2 and the regulatory subunit of phosphoinositide 3-kinase, p85, in a phosphorylation-dependent manner. The evidence suggests that Srcasm may help promote Src family kinase signaling in cells.