N-Myristoyl glycinal diethylacetal strongly inhibited morphological transformation of chick embryo fibroblasts infected with a temperature-sensitive mutant (tsNY68) of Rous sarcoma virus. Myristoylated or nonmyristoylated pp60v-src, which were expressed in tsNY68-infected cells in the absence or presence of the compound, were identified separately by fluorography or immunoblotting analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the [3H]myristate-labeled cell lysate. The results taken together suggest that the blockage of morphological transformation was caused by prevention of protein myristoylation of the transforming protein pp60v-src.