A new mutation, the first one close to the 5'-end of the neurofibromatosis type 1 (NF1) gene, was found when RNA preparations from various cell types of 15 NF1 patients were analysed by reverse transcription and subsequent multiplex polymerase chain reaction. This mutation removes the 84 bp of exon 3 precisely from the cDNA. Genomic Southern blots revealed a larger deletion with breakpoints within the introns flanking exon 3. This mutation suggests that the amino-terminal region of neurofibromin is functionally significant. When using this mutation to distinguish the wild type and mutant alleles, their expression could be analysed in neurofibroma fibroblasts, melanocytes from the unaffected skin, and those from a café-au-lait macule. In all these cell types, the products of both alleles were detected, confirming similar results obtained with a different NF1 gene mutation.