The treatment of cells with staurosporine results in inhibition and less frequently activation of protein kinases, in a cell-type specific manner. In the social amoeba Dictyostelium discoideum, staurosporine induces marked changes in cell morphology affecting growth and development. Here we describe that incubation of D. discoideum growing or starved cells with staurosporine results in a rapid and unexpected tyrosine phosphorylation on two polypeptides of approximately 64 and approximately 62 kDa. These proteins emerge as novel substrates for tyrosine phosphorylation opening up new perspectives for the study of cell signalling in D. discoideum.