The myristyl group makes a critical contribution to the processing, trafficking, and function of myristylated proteins. A series of [omega-125I]iodo-fatty acids was synthesized in order to elucidate the myristyl group's contribution to the membrane association of pp60v-src, the transforming protein of Rous sarcoma virus. In vitro translation of v-src mRNA was employed to monitor incorporation of myristyl analogs into pp60v-src polypeptide. 12-Iodododecanoic, 13-iodotridecanoic, and 14-iodotetradecanoic acids were selectively incorporated in vitro into pp60v-src. One-dimensional peptide analysis confirmed that the analogs were attached to the N terminus of pp60v-src. Upon addition of membranes, the Src proteins modified by these analogs bound to membranes at levels comparable with or slightly less than the myristyl parent. Myristyl analogs were also shown to be incorporated into pp60v-src in vivo. Fractionation of [omega-125I]iodo-fatty acid labeled cells showed that 12-iododecanoic acid, 13-iodotridecanoic acid, and 14-iodotetradecanoic acid modified pp60v-src associated preferentially with the membrane fractions. These results demonstrate that fatty acyl groups one carbon longer or shorter than myristate can be accommodated within the membrane binding site for pp60v-src and illustrate the utility of in vitro systems for predicting analog behavior in vivo. We anticipate that iodinated fatty acids can be used as tools to aid in clarifying the role of the fatty acid in a variety of myristylated molecules.