Abstract A series of derivatives of mitomycin C conjugated with various fatty acids at position 1a was synthesized and the effect of these compounds on protein kinase activities was evaluated. 1a-Docosahexaenoyl mitomycin C (DMMC) selectively inhibited protein tyrosine kinase (PTK) activity in the postnuclear fraction of v- src-transformed NIH 3T3 cells although neither derivatives conjugated with other fatty acids or docosahexaenoic acid or mitomycin C did not. DMMC inhibited the activity of calmodulin-dependent kinase III and protein kinase A very weakly, and only barely affected protein kinase C activity. DMMC also attenuated autophosphorylation of immunoprecipitated p60 v- srcirreversibly. The addition of thiol compounds to the reaction mixture reversed the inhibition by DMMC, suggesting that some thiol moiety of PTK protein might be involved. DMMC also inhibited kinase activity of p210 bcr-abl immunoprecipitated from the lysate of K562 cells. These results indicate that DMMC is a novel inhibitor of PTK.