Antisera were produced against two synthetic peptides having sequences specific for cellular myosin heavy chains from human macrophages (peptide IIA) and bovine brain (peptide IIB). Immunoblots of tissue extracts were made with these antibodies, and they showed that mammalian cells have at least three distinct cellular myosin heavy chain isoforms. Two of the isoforms (MIIB1 and MIIB2) were recognized by antibodies against the peptide IIB, and the other (MIIA) by the anti-peptide IIA antibodies. Polyclonal anti-platelet myosin antibodies recognized the MIIA isoform, but did not recognize MIIB1 or MIIB2. The isoforms were expressed in a tissue-specific pattern; the MIIB1 isoform was found only in brain.