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Background The limitations of conventional sperm analyses have highlighted the need for additional means of evaluating sperm quality. Methods In a study of a cohort of 245 men with known conventional sperm parameters, one-dimensional PAGE was used to monitor protein content and quality in samples from individual ejaculates. Results The sperm protein content varied markedly from sample to another, especially in the high-molecular-weight range. The intensity of the 80–110 kDa bands was correlated with progressive motility (r = 0.15, p = 0.015) and was significantly higher (p = 0.0367) in the group of men with conventional parameters above the World Health Organization’s 2010 reference values than in the group with at least one subnormal parameter (i.e. semen volume, sperm concentration, sperm count per ejaculate, progressive motility, proportion of normal forms or multiple anomaly index below the lower reference value). Using mass spectrometry, the 80–110 kDa bands were found to correspond primarily to three proteins from the flagellum’s fibrous sheath: A-kinase anchor protein 4, A-kinase anchor protein 3, and spermatogenic cell-specific type 1 hexokinase. Conclusion One-dimensional PAGE constitutes a simple, rapid, reliable, inexpensive method for analyzing proteins associated with sperm motility in individual human ejaculates.