One mutation frequently identified in 21-hydroxylase deficiency is the intron 2 splicing mutation, in which the normal polymorphic C or A at nucleotide 655 has been converted to G. Using allele-specific oligonucleotide hybridization, single strand conformational polymorphism analysis, and heteroduplex analyses, we identified 38 individuals from 21 different families who had 2 deleterious mutations. All were homozygous or compound heterozygotes for the splicing mutation. Comparison of the phenotypic features with the molecular genotypes shows phenotypic heterogeneity extending from classical salt-losing 21-hydroxylase deficiency to asymptomatic. Single strand conformational polymorphism analysis followed by DNA sequence analysis revealed numerous sequence variations in intron 2, most commonly at nucleotides 601 and 683. Transient transfection experiments show that the 3'-portion of intron 2 is sufficient to transfer the effect of the 655c/a-->g mutation to a chimeric heterologous gene. Clinical correlations and initial transfection studies suggest that sequence variations at nucleotides 601 and 683 do not correlate with clinical severity or substantially affect splicing. In summary, a single nucleotide change, 655c/a-->g, alters the splice acceptor site at the intron 2/exon 3 boundary. The molecular basis of the phenotypic heterogeneity associated with the mutation remains to be elucidated.