The protein product of the src-related proto-oncogene, fyn, was isolated from IM-9 cells with antibodies specific for the amino-terminal 22 residues of the fyn protein. Peptide mapping demonstrated that the fyn protein was distinct from the closely related c-src and c-fgr proteins. The fyn protein from IM-9 cells incorporated [3H]myristate in vivo and was found to be membrane associated. Phosphoamino acid analysis demonstrated that the fyn protein from IM-9 cells was phosphorylated in vivo predominantly on tyrosine and threonine with only a small amount of phosphoserine detected. the major chymotryptic phosphopeptide of the fyn protein was phosphorylated exclusively on tyrosine. This peptide was specifically precipitated by antibodies directed against a peptide modeled on the closely related carboxy termini of the c-src and fyn proteins. These results provide direct evidence for phosphorylation of tyrosine-531 in the carboxy-terminal chymotryptic peptide of the fyn protein. Phosphorylation of the corresponding site in the closely related c-src protein (tyrosine-527) represses src kinase activity and transforming ability. Loss of the phosphorylation site at tyrosine-531 may similarly contribute to the transforming abilities of carboxy-terminal deletion mutants of the fyn protein.