We identified two homozygous missense mutations in the type II 3β-HSD gene; the first in codon 6 of exon II [CTT (Leu) to TTT (Phe)] in a male infant with pseudohermaphroditism and no apparent salt wasting since neonatal age, and the second in codon 259 of exon IV [ACG (Thr) to AGG (Arg)] in a male pseudohermaphrodite with salt wasting disorder since birth. In vitro expression of the codon 6 mutant enzyme via a mutagenesis study in COS-I cells revealed 25% activity to convert dehydroepiandrosterone (DHEA) to androstenedione (Δ4-A), as compared to 100% activity of wild type, type II 3β-HSD enzyme. In vitro expression of the codon 259 mutant enzyme in COS-I cells revealed no 3β-HSD activity. In addition, our in vitro expression study of a different missense mutation in codon 259 [ACG (Thr) to ATG (Met)] described in another salt waster, whose in vitro study was not previously performed (Human Mol Gen. 1995:4:969), revealed no 3β-HSD activity up to 12 hour incubated COS-I cells, but 10% activity at 24 hour incubated COS-I cells. ACTH stimulated hormonal profiles of the two patients with the novel homozygous mutations in the gene revealed that the 17-hydroxypregnenolone (Δ5-17P)levels and the Δ5-17P to cortisol ratio were exceedingly high as compared to the ACTH stimulated levels in the patients with normal type II 3β-HSD genes as well as those of age, puberty, and sex matched control subjects. However, ACTH stimulatedΔ5-17P to 17-hydroxyprogesterone ratios, DHEA levels, and DHEA toΔ4-A ratios were not consistently elevated. These findings corroborate our earlier observation of the findings in a 32 subject study demonstrating that ACTH stimulated Δ5-17P levels and Δ5-17P to F ratios are consistently useful criteria to identify the patients with deleterious mutant type II 3β-HSD genes, while ACTH stimulated Δ5-17P to 17-OHP ratios, DHEA levels, and DHEA to Δ4-A ratios are not consistently useful to distinguish between the patients with or without deleterious type II 3β-HSD gene mutations. These findings help to further the understanding of the type II 3β-HSD gene function, the molecular basis of 3β-HSD deficiency CAH, and the hormonal criteria for accurate diagnosis of bonafide 3β-HSD deficiency CAH based on genotypic proof.