The EVI2B gene is one of three genes embedded in intron 27b of the neurofibromatosis type 1 (NF1; M. Recklinghausen) gene, which are transcribed in the direction opposite that of the NF1 gene. The function of EVI2B and its relation to NF1 symptoms is unknown. Here, the amounts of NF1 and EVI2B mRNA were investigated in detail in cells involved in NF1 manifestations as café-au-lait macules and neurofibromas. These investigations showed that aside from the NF1 gene, EVI2B is involved in melanocyte and keratinocyte differentiation. Whereas in NF1 melanocytes from café-au-lait macules, EVI2B expression was not altered, in fibroblast-like cells derived from neurofibromas, an increased level of EVI2B mRNA was found. We investigated whether this increase was attributable to an influence of NF1 gene expression on the expression of the EVI2B gene, as suggested by the fact that the EVI2B primary transcript is antisense to the NF1 primary transcript. Investigations of cells derived from patients with different amounts of NF1 pre-mRNA showed no correlation between the amount of NF1 pre-mRNA and the increased level of EVI2B mRNA.