New insights into the gene orders, structures, evolution, and functions of streptomycin (Sm) biosynthetic genes (str) were gained via hybridization studies, determination of nucleotide sequences, and measurement of expression in the str gene clusters of Streptomyces griseus and S. glaucescens. Both str clusters showed considerable divergence in macro and micro structure. Genes putatively involved in pathways leading to the (dihydro-)streptose and N-methyl-L-glucosamine moieties of Sm were identified. Additional regulatory elements, such as gene strS and conserved TTA codons in the N-terminal sections of reading frames, are reported. Evidences for the involvement of physiological state, signal transduction, and activators in the control of Sm production are presented.