In an attempt to identify genetic lesions contributing to human growth disorders, we evaluated a prospectively recruited group of children with growth failure for mutations in the insulin-like growth factor-I (IGF-I) gene. Two complementary approaches were used: Southern blot analysis to examine the large scale organization of the gene, and a solution hybridization, nuclease protection assay to identify small alterations, such as point mutations. From a total of 61 subjects studied, 52 had no organic basis for their short stature. Analysis of chromosomal DNA from these individuals failed to reveal any variation in the IGF-I gene except for a HindIII site polymorphism which maps near the 3' end of the last IGF-I exon. No single nucleotide substitutions were found within IGF-I-coding regions. Since the frequency of the length polymorphism was the same for both normal-sized and short individuals, it is unlikely to be associated with growth abnormalities. Our results suggest that there is minimal DNA sequence variability in the human IGF-I gene and that mutations in IGF-I exons are infrequent causes of growth failure.