More than 90% of all cases of congenital adrenal hyperplasia result from steroid 21-hydroxylase (CYP21) gene mutations. Around 95% of these are either gene deletions or any of nine sequence aberrations that have been transferred from the nearby pseudogene through apparent gene conversions. One such recurrent pseudogene-derived mutation is Cluster E6, a combination of three amino acid substitutions in exon 6: I236N, V237E, and M239K. Cluster E6 is associated with the most severe, salt-wasting form of congenital adrenal hyperplasia. We studied the functional consequences of each missense mutation individually as well as the combined effect of the three mutations comprising Cluster E6. V237E abolished enzyme function and is thus a null mutation, whereas very low but measurable activity remained for I236N. M239K, on the other hand, had no effect on enzyme activity and consequently does not contribute to the disease. Although no allele has been reported yet to contain only one or two missense mutations of Cluster E6, it is a well-known feature of CYP21 that it can harbor many different combinations of pseudogene-derived mutations. The exclusion of M239K as a disease-causing mutation is thus relevant when designing protocols for genetic diagnostics.