The streptavidin gene from Streptomyces avidinii was expressed in E. coli as a non-fusion protein and as a glutathione S-transferase fusion protein. The streptavidin protein accumulated primarily in the inclusion bodies and did not alter cell growth. In contrast, the glutathione-S-transferase-streptavidin fusion protein was soluble. Nondenaturing polyacrylamide gel electrophoresis indicated that the chimeric glutathione-S-transferase-streptavidin protein was present mostly as a monomer, with some detectable polymeric forms. Cells grown in the presence of [H-3]-biotin had label specifically associated with the expressed glutathione-S-transferase-streptavidin fusion protein, indicating this protein bound biotin in vivo. The majority of the radiolabeled biotin was associated with polymeric forms of the glutathione-S-transferase-streptavidin protein. The growth rates of biotin auxotrophs of E. coli growing in biotin-deficient media were substantially decreased by the expression of the glutathione-S-transferase-streptavidin gene. The decreased growth rate correlated with a decrease in acetyl-CoA carboxylase activity.