The incidence of impaired testicular descent (cryptorchidism) is high; 1-2% of boys at the age of 3 mo are diagnosed for this condition in western countries. Recent data on mice with targeted disruption of the Insl3/relaxin-like factor (RLF) gene proposed that this factor plays a role in testicular descent in fetal life. Male RLF-/- mice exhibit bilateral cryptorchidism due to developmental abnormalities of the gubernaculum, associated with abnormal spermatogenesis and infertility. In the present study, we have sequenced the promoter region and both exons of the RLF gene in a cohort of 30 boys, seven of whom presented with a possible familial form of cryptorchidism and 23 with sporadic cryptorchidism. One of the nucleotide substitutions detected, G to A at position 178, predicted amino acid change. The mutation was localized to the C-peptide region, resulting in an alanine to threonine change and therefore classified as a conservative mutation. Four of the 30 cases analyzed were homozygous (13%), and 15 were heterozygous for the mutation (50%). However, the same mutation was also found in a control group of 89 men; 10% of them were homozygous, and 39% were heterozygous. Our results indicate that mutations in the RLF gene are not a common reason for cryptorchidism and that the common G178A polymorphism has no apparent relationship with this condition.