A steroid-binding protein, identified in the supernatant generated when membrane vesicles of Pseudomonas testosteroni are produced and harvested by centrifugation, has been purified 49-fold to homogeneity. It has a molecular weight of 30 000-35 000 and it specifically binds the C19 steroids dihydrotestosterone, testosterone, and androstenedione. It is a basic protein with an isoelectric point at pH 7.3. Binding of testosterone exhibited normal saturation kinetics with an affinity constant, Kd, of 3.9 X 10(-8) M. Binding was inhibited by divalent cations, but the sulfhydryl reagents dithiothreitol and mercaptoethanol did not affect activity. It is suggested that this and other membrane-associated steroid-binding proteins concentrate the steroid at the membrane surface before it is transported into the cytoplasm of P. testosteroni.