Deficiency of adrenal steroid 21-hydroxylase is the most common form of congenital adrenal hyperplasia and it is considered to be responsible for 90% of the disease. This paper describes for the first time the CYP21B mutation profile in Brazilian patients. We genotyped 41 families with at least one individual affected with the classical form of the 21-hydroxylase deficiency, representing 74 unrelated alleles. In order to characterize different disease-causing alleles, genotyping was performed by Southern blot analysis with three restriction enzymes, allele-specific oligonucleotide hybridization, and allele-specific PCR. Different alleles were distinguished by TaqI C4B RFLP, gene duplications or deletions of either CYP21A + C4B or CYP21B + C4B, large gene conversions and eight mutations that might have been introduced into CYP21B from CYP21A by microconversion events. At least one mutation was detected in 24 different disease-causing alleles, which represents about 85% of the affected alleles in those families. The frequency of the 30 kb deletion of CYP21B was lower than that described for Caucasians. The mutation Sp2 showed the highest frequency (24.65%) and was present mainly in salt-wasting patients, although it was also detected in some patients with the simple virilizing form of the disease. Conversely, I172N showed a frequency of 18.91% and was found mostly in patients affected with the simple virilizing form of the disease. Five other mutations were determined at low frequency, but CL6 was not found in any of the tested alleles.