17 beta-Hydroxysteroid dehydrogenase type 3 (17 beta-HSD3) is expressed almost exclusively in the testis and converts Delta 4-androstene-3,17-dione to testosterone. Mutations in. the HSD17B3 gene causing 17 beta-HSD3 deficiency are responsible for a rare recessive form of 46, XY Disorders of Sex Development (46, XY DSD). We report novel cases of Tunisian patients with 17 beta-HSD3 deficiency due to previously reported mutations, i.e. p.C206X and p.G133R, as well as a case with the novel compound heterozygous mutations p.C206X and p.Q176P. Moreover, the previously reported polymorphism p.G289S was identified in a heterozygous state in combination with a novel non-coding variant c.54G >T, also in a heterozygous state, in a male patient presenting with micropenis and low testosterone levels. The identification of four different mutations in a cohort of eight patients confirms the generally observed genetic heterogeneity of 17 beta-HSD3 deficiency. Nevertheless, analysis of DNA from 272 randomly selected healthy controls from the same geographic area (region of Sfax) revealed a high carrier frequency for the p.C206X mutation of approximately 1 in 40. Genotype reconstruction of the affected pedigree members revealed that all p. C206X mutation carriers harbored the same haplotype, indicating inheritance of the mutation from a common ancestor. Thus, the identification of a founder effect and the elevated carrier frequency of the p. C206X mutation emphasize the importance to consider this mutation in the diagnosis and genetic counseling of affected 17 beta-HSD3 deficiency pedigrees in Tunisia. (C) 2016 Elsevier Ltd. All rights reserved.