In order to explain the heterogeneity of Ca antigen (Ca) expression observed previously on human malignant cells, a relationship of antigen content with events in the cell cycle was studied by multiparameter flow cytometry on four continuous human cell lines, HeLaS3, C4l, HT29, and T24, grown exponentially in culture. In the four cell lines studied, there was an increase in Ca expression during the G1 and G2 + M phases whereas, during the S phase, the Ca expression was relatively constant. Although the level of Ca varied among the four cell lines, the mean amount of Ca expressed during the G2 + M phase was from 58 to 94% higher than the mean for the G1 phase. The G1 and G2 + M cell populations also displayed considerably greater variability of Ca content when compared with cells in the S phase. The level of Ca among cell lines appeared to have an inverse relationship to DNA content expressed as DNA index. Ca expression appeared to be related to RNA content and hence, presumably, ribosomal content, across the cell cycle. This study suggests that some of the heterogeneity of Ca expression in human cancer cells may be related to cell cycle events.