The autophosphorylating protein, Ptk, of the bacterium Acinetobacter johnsonii was overproduced, purified to homogeneity and assayed for ATP binding by using the nucleotide analog 5'-p-fluorosulfonylbenzoyl adenosine. The ATP binding site of this bacterial autophosphorylating protein was found to be different from that generally used by eukaryotic protein kinases. It consists of two amino acid sequences that closely resemble the Walker motifs A and B. This observation was confirmed by site-directed mutagenesis experiments which showed, in addition, that the ATP molecule bound to these motifs is effectively employed by the bacterial protein to autophosphorylate on tyrosine. It is concluded that even though the overall autophosphorylation reaction is similar in eukaryotic and prokaryotic proteins, the mechanism involved is likely different.