The neurofibromatosis type 1 (NF1) gene encodes a tumor suppressor protein, termed neurofibromin, which is expressed predominantly in neurons, Schwann cells, oligodendrocytes, and leukocytes. There are at least three isoforms of neurofibromin produced by the alternative use of exons 23a and 48a. Previously we described the identification of an NF1 mRNA isoform containing an additional 54 nucleotides from exon 48a (type 3 NF1 ) in human skeletal, cardiac and smooth muscle tissues by reverse‐transcribed (RT)‐PCR. To extend our initial observations, we have produced high titer chicken IgY antibodies which specifically recognize this muscle‐specific neurofibromin isoform. An NF1 cDNA was generated containing human exon 48a sequences and expressed as a fusion protein in bacteria. The muscle‐specific neurofibromin antibodies detected this exon 48a fusion protein by Western immunoblotting. Immunoprecipitation using these type 3 neurofibromin antibodies also specifically detected a 250 kDa protein in human and rat muscle tissues. Type 3 neurofibromin was found in rat heart and muscle, but not in live, brain, kidney or spleen with levels of expression declining after postnatal day 7. Expression of total NF1 RNA during rat embryonic development was detected at high levels in E15 heart, tongue, and limb bud. In addition, using type 2 neurofibromin‐specific antibodies, the existence of a fourth isoform of neurofibromin (type 4 neurofibromin) containing both exon 23a and 48a sequences was demonstrated in rat heart muscle tissues. The identification of two muscle‐specific isoforms of neurofibromin expands our definition of this important tumor suppressor protein and suggests additional roles for neurofibromin in muscle development and differentiation. © 1995 Wiley‐Liss, Inc.