A gene pknA, coding for an eukaryotic-type protein Ser/Thr kinase, was cloned from the Streptomyces coelicolor A3(2) chromosome. The PknA protein kinase, containing the C-terminal eukaryotic-type kinase domain with an N-terminal extension, was expressed in Escherichia coli and Streptomyces lividans. The affinity purified MBP-PknA fusion protein was assayed for kinase activity that showed its ability to autophosphorylate in vitro in the presence of [gamma-P-32]ATP. The activity was Mn2+-dependent, The preautophosphorylated kinase phosphorylated at least two proteins (sizes 30 and 32 kDa) in the S. coelicolor J1501 cell-fi ee extracts of all developmental stages. The larger of them was also phosphorylated in vitro by an endogenous protein kinase in late stages extracts, but not earlier. Although Mn2+-dependent protein phosphorylation has previously been described In Streptomyces, this is the first report of a gene encoding such an enzyme in this genus.