The amino-terminal region of the human sex steroid-binding protein of plasma (SBP or SHBG) containing K134 and M139 was found to represent part of the steroid-binding site. This was accomplished by constructing and expressing site-directed mutants having the following replacements: M139L, M139K, M139S, K134A, H235S, and Y57F. The results indicated that M139L and H235S were fully-active, K134A and Y57F were 50 and 67% active, M139K was 7% active, and M139S was inactive. These results support affinity-labeling data indicating that both K134 and M139 are located in or near the site, and suggest that Y57 may play a role in steroid binding. The fully active H235S mutant reveals that H235 is not involved in the steroid-binding process.