In recent years, partitioning in aqueous two-phase systems has been developed as a mild separation procedure of high selectivity for studying cell surface properties. This work was undertaken to study the surface heterogeneity of bull and ram sperm cells by using centrifugal counter-current distribution in dextran-poly(ethylene glycol) two-phase systems. The revealed heterogeneity appears to be associated with different viability states of the fractionated cells. Various strands of evidence support this conclusion: (a) assessment of viability by fluorescence probes showed a different enrichment of live cells in the different fractions tested, dead spermatozoa showing enhanced affinity to the dextran-rich phase; (b) freezing or thermic shock of sperm cells, known methods of promoting loss of viability, provoked an increase in the number of cells with enhanced affinity to this phase; (c) the same effect was observed when sperm cells were obtained from ejaculates after longer periods of abstinence. The results presented here strongly suggest that loss of semen viability results in a decreased hydrophobicity of the cell surface. Thus this may be suitable for detecting sperm surface changes and to separate spermatozoa populations enriched in some functional and/or structural surface features.