The Src family consists of nine related tyrosine protein kinases with a common domain structure, including a myristylated N-terminal glycine residue. In this report, we identify cysteine residues within the N-terminal region of the Src family member Fyn which serve as sites for palmitylation. To facilitate detection of protein fatty acylation, p59fyn was overexpressed in COS cells and incubated with radioiodinated fatty acid analogs of myristate (IC13) or palmitate (IC16). Incorporation of both fatty acids into p59fyn was readily observed. Acylation with the palmitate analog was prevented when Gly-2 was mutated to alanine, implying that N-myristylation is required for palmitylation, and when either Cys-3 or Cys-6 was mutated to serine. Palmitylation was shown to alter the distribution of p59fyn between membrane-bound and soluble fractions. In contrast, no incorporation of the palmitate analog into pp60v-src, which lacks N-terminal cysteine residues, was observed. Mutation of Ser-3 of Src to cysteine, but not Ser-6, resulted in incorporation of the palmitate analog. These results serve to delineate sequence elements important for dual acylation of proteins, and further illustrate the utility of radioiodinated fatty acid analogs for studies of protein fatty acid acylation.