Three different new mutations were found after CYP21 gene sequencing in three unrelated patients with the classical form of the 21-hydroxylase deficiency. These mutations were also screened in their affected relatives. In one patient and her brother, both affected with the simple virilizing form and in their aunt, with the nonclassical form, an AG>GG transition was found in the acceptor site of intron 2. In another patient with the salt wasting form, we found a 1003 1004 insA, in exon 4, that altered the reading frame and created a stop codon in codon 297. In the third patient and his sister, we found a C>T transition in codon 408. This transition led to the substitution of arginine by cysteine (R408C) in a conserved region where arginine is conserved in at least four different species. These siblings with the R408C mutation, both affected with the salt wasting form, have the IVS2-13A/C>G mutation in the other allele, suggesting that the R408C should lead to complete impairment of enzymatic activity. To rule out the possibility of polymorphism, R408C was screened through allele specific PCR, and it was not found in 100 normal alleles. The screening of these three new mutations by allele-specific PCR or enzymatic restriction in 212 CAH patients disclosed their presence in 2.3% (9/387) of the alleles. All three new mutations were found in compound heterozygous state with previously known mutations. Microsatellite studies, using markers flanking CYP21 gene, revealed that each new mutation presents the same haplotype, suggesting a gene founder effect, similar to what was previously observed with the G424S mutation also described in our population. Although microconversion events are the main cause of mutations in the CYP21 gene, random mutations with a common origin can also be the cause of 21-hydroxylase deficiency.