The neurofibromatosis 1 (NF1) gene encodes a cytoplasmic protein with structural and functional homology to GTPase activating proteins (GAPs) for p21-ras. Double-labeling immunofluorescence experiments using neurofibromin antibodies and in vitro microtubule assembly have demonstrated that the NF1 gene product, neurofibromin, interacts with cytoplasmic microtubules. The region critical for this interaction was shown to reside within the NF1-GAP-related domain (NF1GRD). Examination of the NFIGRD reveals a number of residues that are highly conserved amongst all GAP molecules. Mutational analysis of a representative number of these conserved residues demonstrated differential effects on NF1GRD p21-ras GAP activity. In this study, we examined the effect of these selected NF1GRD mutations on the ability of neurofibromin to associate with microtubules. Mutations at residues R1391, P1400 and K1423 disrupted microtubule association. In contrast, mutations at residues E1264, Q1426 and the insertion of exon 23a, critical for p21-ras regulation, did not impair microtubule association. These results demonstrate that some residues important for p21-ras regulation are also required for microtubule binding while other residues within the NF1GRD differentially affect p21-ras regulation and microtubule association.