Neurofibromatosis 1 (NF1) is a common genetic disorder characterized by abnormalities of tissues derived from the neural crest. To define germ-line mutations in the NF1 gene, we studied 20 patients with familial or sporadic cases of NF1 diagnosed clinically and one patient with only café-au-lait spots and no other diagnostic criteria. A protein truncation assay identified abnormal polypeptides synthesized in vitro from five RT-PCR products that represented the entire NF1 coding region. Truncated polypeptides were observed in 14 individuals. The mutations responsible for the generation of abnormal polypeptides were characterized by DNA sequencing. Thirteen previously unpublished mutations were characterized in the 14 individuals. The mutation 2027insC was observed in two unrelated individuals; the other 12 mutations were unique. The sequence changes included seven nonsense and four frameshift mutations that created premature translation termination signals, and two large in-frame deletions that led to the synthesis of truncated polypeptides. One of the mutations was found in the child with a single clinical diagnostic criterion, providing her with a presumptive diagnosis of NF1. Our results confirm that truncating mutations are frequent in both familial and sporadic NF1 cases. The identification of mutations in 14 of 21 individuals studied (67%) suggests that the use of protein truncation assays will rapidly accelerate the rate of identification of NF1 mutations. Because we scanned the entire NF1 coding region in each individual, the distribution of NF1 truncating mutations was discerned for the first time. The mutations were relatively evenly distributed throughout the coding region with no evidence for clustering.