We determined the isoforms of tropomyosin expressed and the level of tropomyosin phosphorylation in donor, end-stage failing and hypertrophic obstructive cardiomyopathy samples of human heart muscle. Western blots and isoform-specific antibodies showed that α-tropomyosin was the only significant isoform expressed and that tropomyosin was 25-30 % phosphorylated at serine 283. Mass spectrometry confirmed directly that α-tropomyosin made up over 95 % of tropomyosin but also indicated the presence of up to 4 % κ-tropomyosin and much smaller amounts of β-, γ- and smooth β-tropomyosin and about 26 % phosphorylation. Neither the isoform distribution nor the level of phosphorylation changed significantly in the pathological heart muscle samples. © 2013 Springer Science+Business Media Dordrecht.