Natural multiple pregnancy in women leading to dizygotic (DZ) twins is familial and varies across racial groups, suggesting a genetic predisposition. Mothers of DZ twins have a higher incidence of spontaneous multiple ovulation and elevated FSH concentrations. FSH release is controlled by feedback of inhibin peptides from the ovary, and immunization against inhibin α-subunit results in an increased ovulation rate in animals. The inhibin α-subunit is therefore a candidate gene for mutations that may increase the frequency of DZ twinning. Restriction digests of a PCR product from exon 1 with the enzyme SpeI detects a C/T polymorphism at bp 128 with two alleles of 447 and 323/124 bp. The polymorphism was typed in 1125 individuals from 326 pedigrees with 717 mothers of spontaneous DZ twins. The α-inhibin locus mapped within 3 centimorgans of D2S164, and linkage with DZ twinning was excluded [decimal log odds ratio (LOD) score, −2.81 at θ = 0]. There was complete exclusion of linkage (LOD, less than −2) of a gene conferring relative risk 1.8 (λs, >1.8) across the chromosome, except at the p-terminus region and a small peak (maximum LOD score, 0.6) in the region of D2S151-D2S326. Analysis using either recessive or dominant models excluded linkage with DZ twinning in this population (LOD score, less than −2.5) across chromosome 2. We conclude that dizygotic twinning is not linked to variation in the α-inhibin locus. The results also suggest that mutations in other candidates on chromosome 2, including the receptor for FSH and the βB-inhibin subunit (INHBB) cannot be major contributors to risk for DZ twinning.