The activity of the c-Src protein is increased during cell-cycle stage G1 in fibroblasts stimulated with certain growth factors, and at the G2/M transition, but little is known about Src substrates in these circumstances. In contrast, cells transformed with activated Src contain many tyrosine-phosphorylated proteins. We compared the phosphotyrosine content of growing and mitotically arrested Src-transformed cells. We report here that although phosphorylation of most proteins was unchanged during mitosis, phosphorylation of one of about 68K was greatly enhanced. The p68 was physically associated with activated c-Src, and it bound to the SH3 domain of c-Src in vitro. Tyrosine-phosphorylated p68 was also present in mitotic extracts of normal cells, suggesting that its phosphorylation was not just a consequence of transformation. Purification and microsequencing of p68 showed that it was related to the previously described GAP-associated protein p62 (ref. 6).