The association of cytochrome c oxidase negative fibres (COX-negative) and ragged-red fibres (RR-fibres) with work related trapezius myalgia has been proposed. Hitherto studies have been small or without control groups. The aim of the present study was to investigate the prevalences of RR-fibres and COX-negative fibres in female cleaners with (n=25) and without (n=23) trapezius myalgia and in clinically healthy female teachers (n=21). The cleaners did mainly floor cleaning requiring monotonous loading on the trapezius muscle. A questionnaire covering background data and aspects of pain (prevalence, duration, intensity and influence on daily living) was answered. Biopsies were obtained from the trapezius muscle by an open surgical technique. The three groups did not differ in prevalence of COX-negative or COX-superpositive (i.e. type-I fibres with extremely strong brownish reaction in both the COX and SDH/COX stainings) fibres. The prevalence of COX-negative fibres was age dependent. Two subgroups of RR-fibres were present when stained for COX, COX-negative (73%) and COX-superpositive (26%) fibres. Forty-two percent of the COX-negative fibres were RR-fibres and 79% of the COX-superpositive were RR-fibres. A significantly (P=0.002) higher proportion of the COX-superpositive fibres in the cleaners were RR-fibres compared to the teachers. Multivariate regression analysis revealed that age, occupation as cleaner and a tender point in the trapezius were significantly associated with increased prevalences of RR-fibres, a cleaner with a tender point had a 4.35 higher prevalence of RR-fibres compared to a teacher without a tender point. No correlations between other pain related variables and prevalence of RR-fibres were noted. In conclusion, RR-fibres but not COX-negative or COX-superpositive fibres were correlated with cleaning work tasks and with a tender point in the trapezius.