Enolase, lactate dehydrogenase, and phosphoglycerate mutase have previously been found to contain phosphotyrosine in fibroblasts transformed by Rous sarcoma virus, which encodes a tyrosine-specific protein kinase. However, these phosphorylations are not stoichiometric, and their significance for any aspect of the transformed phenotype is unknown. We show here that enolase and lactate dehydrogenase are each phosphorylated chiefly at a single tyrosine in Rous sarcoma virus-transformed cells. The purified enzymes can also be phosphorylated at the same tyrosine in vitro when incubated with an immunoprecipitated retroviral transforming protein having associated tyrosine protein kinase activity. The phosphorylated tyrosine in lactate dehydrogenase is amino acid 238. The phosphorylated tyrosine in enolase lies in a sequence homologous to that surrounding histidine 43 in yeast enolase. Although the phosphorylated sequence in lactate dehydrogenase shows some homology to those sequences surrounding phosphotyrosines found in retroviral transforming proteins, the phosphorylated sequence in enolase is quite different.