We studied the NF1 gene in 93 unrelated patients with neurofibromatosis type 1, focusing the analysis on four exons that contain the highest number of possible mutations occurring at CpG sites. We used denaturing gradient gel electrophoresis to analyse exons 16, 28, 29 and 49, which contain 45 (25%) of the 183 possible mutations that could occur at the 120 CpG dinucleotides of the coding sequence. Six different mutations were identified, five of which are novel: two truncating mutations, W1810X and 5448insG, located in exon 29; two splice defects leading to exon 29 skipping, 5206‐2A>G and 5546G>A; and one missense mutation, L844F, located in exon 16. The already described R1748X mutation located in exon 29 was found in two unrelated patients. The 5546G>A and R1748X mutations are located at CpG sites, whereas the W1810X involves a CpNpG site. Four novel polymorphisms, which may be helpful for family studies, were also identified. Overall, all but one mutations were found in exon 29, a result which suggests that all the CpG sites of the NF1 coding sequence do not have the same mutability, and that exon 29, the most CpG‐rich exon, contains mutational hotspots associated with NF1. Hum Mutat 16:274–275, 2000. © 2000 Wiley‐Liss, Inc.