Heart muscle has been considered to contain predominantly α-Tm (TPM1 gene). However this has not been investigated with tools that can clearly distinguish all tropomyosin isoforms. We initially used LC/MS to characterise the isoforms present in Tm purified from human heart muscle. Mass spectroscopy of Tm purified from human donor heart showed two dominant species, a 32754 Da peak, corresponding to acetylated α-Tm and a 32867 Da peak. The second peak was putatively identified by its mass as γ-Tm. These identities were confirmed by comparison with pure recombinant α and γ-skeletal tropomyosin expressed in the baculovirus/sf9 system (supplied by Dr Kristen Nowak, University Western Australia). The novel Tm is slow-skeletal Tm from the TPM3 gene, also known as γ-skeletal tropomyosin, (theoretical mass 32859.0 Da). Only very low levels (<5%) of κ Tm or of phosphorylated Tm were detected. Analysis of samples from failing heart muscle and in myectomy samples showed significantly reduced γ-Tm levels.The identity of the isoforms characterised in human heart was confirmed by urea SDS-PAGE, which can separate the two species, and the use of isoform-specific antibodies. Using the pure recombinant Tms, we demonstrated that monoclonal antibody CG3 was specific for the γ isoform, CH291 was specific for the α isoform and CH1 did not distinguish between the two isoforms. Western blotting with these specific antibodies confirmed significant quantities of γ-Tm and α-Tm in both human and mouse hearts. In mice, the level of γ-Tm depends on the strain, being around 50% in many strains, but low in FVB/N mice.This discovery is functionally significant as in vitro motility assays using pure γ-Tm and α-Tm shows significant differences between the isoforms.