Three patients with neurofibromatosis 1 (NF1) were operated for congenital pseudarthrosis (PA) of the tibia. Three non-NF1 patients served as reference. Both NF1 mRNA and protein were detected in the PAs and in rows of osteoblasts and numerous osteoclasts next to the NF1-related PA arguing against inactivation of both NF1 alleles in the resident cells. Analyses on mesenchymal stem cells (MSCs) cultured from the red bone marrow of 1) next to PA of the affected NF1 tibiae, 2) the non-affected NF1 iliac crest of the same patients, and from 3) non-NF1 bone marrow demonstrated that the potential to form bone in vitro was the lowest in cells from the affected NF1-tibiae. The latter cells also displayed reduced levels of NF1 mRNA and protein, and upregulated phosphorylated p44/42 MAPK levels, consistent with an upregulated Ras-pathway. An exhaustive NF1 gene analysis detected constitutional mutation in each case, but no second hits or loss of heterozygosity were found. However, one patient displayed a mutation resulting in two potential active splice sites ultimately affecting exon 6. Interestingly, only one of the respective transcripts was detected in cells from the iliac crest, but two novel transcripts were detected in MSCs cultured from site next to PA. This finding may identify a novel mechanism how a single NF1 gene mutation may exert distinct effects on separate anatomical locations. The molecular pathogenesis of NF1-related PA apparently may not be entirely explained by second mutations or loss of heterozygosity of the NF1 gene. © 2008 Elsevier Inc. All rights reserved.