The severe combined immunodeficiencies (SCID) are a heterogeneous group of conditions arising from a variety of molecular defects. The X-linked form of SCID (X-SCID) is caused by defects in the common gamma chain (γc), and is characterized by a T–B+NK− immunophenotype. This lymphocyte profile is seen in an autosomal recessive form of SCID caused by mutations in the JAK3 molecule. Thus, X-SCID and JAK3-deficient SCID are clinically and immunologically indistinguishable. Knowledge of the precise molecular defect is essential for antenatal diagnosis, carrier testing and for treatment using somatic gene therapy. To identify the molecular defect in children presenting with a T–B+NK− form of SCID, we have developed rapid assays based on flow cytometric analysis of γc, immunoblotting for JAK3 and γc, and detection of interleukin-2 (IL-2)-induced tyrosine phosphorylation of JAK3. Sixteen T–B+NK− SCID patients from 15 families were examined. Nine had no detectable γc, four had abnormal γc expression and no IL-2-induced JAK3 tyrosine phosphorylation, and one had normal γc expression but no IL-2-induced JAK3 tyrosine phosphorylation, although JAK3 was present. All these patients had mutations identified in their γc gene. Two patients exhibited normal γc expression, but JAK3 was not detected by immunoblotting and these patients were confirmed as having JAK3 gene mutations. Thus, these protein-based assays have led to rapid molecular diagnoses in T–B+ SCID that have subsequently been confirmed by genetic analysis.