Short stature is associated with abnormalities of the growth hormone (GH) axis. We hypothesized that an aberrant, less functional, GH molecule due to a growth hormone gene (GH-I) mutation may lead to short stature due to abnormal secretory dynamics or abnormal interaction with the GH receptor. Genomic DNAs from patients (n = 130) with short stature (height < 2 S.D. below mean for age) and normal statured subjects (n = 108) were screened for abnormalities in the coding region of GH-I. The coding region was amplified from genomic DNA by polymerase chain reaction, and assayed by solution hybridization/nuclease protection. Five patients with short stature were identified with point mutations of GH-I in exon 2 compared to none in the normal statured group (p = 0.048). On Southern blotting, the mutation in all five patients created a new HincII restriction site, as evidenced by disappearance of the expected 6.6 Kb band in 3 of 5 patients and appearance of a new 4.5 Kb band in all 5 patients. The exact nature of these mutations will be characterized and studied for their biological activity. The assay described here identifies GH-I mutations in children with short stature which may impact GH function.