Classical CAH due to 3β-HSD deficiency is associated with distinct point mutations identified only in the type II gene, and stop codon homozygous mutation has been detected in only one family. A small number of affected females have been reported, presenting different clinical phenotypes, including normal external genitalia, mild virilization, amenorrhea and even an asymptomatic form. We report a white female child who was born from consanguineous parents originating from Chile. At birth hyperpigmentation of normal external female genitalia was noticed. At 60 days she developed hyponatremia and hypercalemia, which improved after gluco and mineralocorticoid therapy. At 20 months of age the diagnosis of 3β-HSD deficiency was achieved based on elevated 17-hydroxypregnenolone (17Preg=79.5 ng/ml; normal values=0.1-2.1) and 17-hydroxypregnenolone/17-hydroxyprogesterone (17OHP) ratio (24.8; normal values=0.4-2.5) in basal conditions and after exogenous ACTH (17Preg=82.8, normal=0.5-7.3; 17Preg/17OHP=12.9, normal=0.5-3.3). Her karyotype is 46,XX. Genomic DNA was extracted from peripheral blood in filter paper, amplified by PCR, and screened for mutations by denaturing gradient gel electrophoresis. PCR produts of altered mobility were sequenced directly by the di-deoxy method. A novel mutation in type II 3β-HSD gene was found in codon 135, leading to a homozygous substitution GAA (Glu) to stop codon (TAA). Her parents are heterozygous for the mutation. The E135X mutation would lead to a predicted truncated protein. The present homozygous nonsense mutation in type II 3βHSD gene indicate a novel genetic alteration responsible for 3β-HSD deficiency in a female child with salt-losing CAH.