Extraocular muscles contain both fast-twitch and multiply-innervated, tonic-contracting fibres. In rat, these fibres collectively express numerous myosin heavy chain isoforms including fast-type embryonic and neonatal, adult slow twitch type I and fast twitch type II, and a fast isoform unique to extraocular muscle. Immunocytochemical and Western blotting results are presented which suggest that, in rabbit, an additional species, the alpha-cardiac myosin heavy chain, is present. The immunoreactive species is found in all rabbit extraocular muscles and in the extraocular muscles is expressed in almost all fibres which do not contain a fast myosin heavy chain. Positive identification of this isoform as the alpha-cardiac myosin heavy chain was obtained by sequencing a cloned PCR product derived from extraocular muscle mRNA unique to the 3'-end of rabbit alpha-cardiac myosin heavy chain mRNA. This is the first unequivocal demonstration of alpha-cardiac myosin heavy chain expression in extraocular muscle.