The entire nucleotide sequence of a cloned 2568-bp PstI fragment from the genome of Streptococcus equisimilis H46A encoding the streptokinase gene (skc) has been determined. The longest open reading frame comprises 1320 bp which code for streptokinase. The protein is synthesized with a 26-amino acid residue N-terminal extension having properties characteristic of a signal peptide. Comparison of the deduced amino acid sequence with the available amino acid sequence of a commercial streptokinase reveals minor primary structure differences. The nucleotide sequencing of skc does not support the hypothesis that the gene has evolved by duplication and fusion, as suggested by internal twofold amino acid homologies of its product. Furthermore, the skc gene sequence shows no extended regions homologous to the staphylokinase gene. Upstream from the skc gene, the putative skc promoter and the ribosome-binding site sequence have been identified; downstream from the coding region, inverted repeat sequences thought to function as transcription terminators have been detected.