1. The function of a Y human chromosomal DNA sequence was evaluated. The Y-5 probe was isolated from a flow-sorted chromosome library and detects Y-specific sequences. 2. The Y-5 probe and other Y-specific probes were used to analyze an XX male patient without ambiguous genitalia. 3. DNA sequences from the short arm of the chromosome Y that were detected with pDP1007 and pDP105 in the patient's genome explain the testis differentiation observed in this case. 4. Failure of the patient's DNA to hybridize to the Y-5 probe shows that the primitive gonads can differentiate into testes even in the absence of this chromosome region. In contrast, a gene controlling spermatogenesis may exist in this region because the patient is azoospermic.