Although a novel brain-specific fatty acid-binding protein (B-FABP) was recently cloned, the identity of a second fatty acid-binding protein detected with antibodies to the heart (H-FABP) has not been clearly resolved. The present investigation, using matrix-assisted laser desorption mass spectrometry, showed that this protein was a form of H-FABP whose N-terminal amino acid was neither methionine nor was it acetylated. Furthermore, isoelectric focusing revealed two major isoforms, a major band pl 7.4 and a minor band pl 6.4, in a distribution pattern opposite to that observed for H- FABP in the heart. Tryptic peptide mass maps of the in-gel digested SDS polyacrylamide gel electrophoresis protein bands showed that the two isoforms differed only in a single peptide corresponding to residues 97-106 of the heart H-FABP sequence. This peptide had an [M + H]+ ion of either 1205.62 (pl 7.4) or 1206.53 (pl 6.4), consistent with a single amino acid substitution, Asp98 or Asn98. Whereas it is well established that both H- FABP and B-FABP interact with polyunsaturated fatty acids, we showed that they also significantly alter plasma membrane cholesterol dynamics in a manner opposite to that of another brain lipid-binding protein, sterol carrier protein-2. In summary, the data demonstrated for the first time that the H-FABP from brain, while nearly identical to H-FABP from heart, differed significantly in isoform distribution and in amino terminal structure from heart H-FABP. This suggests that the brain and heart H-FABP may not necessarily function identically in these tissues.