Endogenous cytokinins were quantified in synchronized Chlorella minutissima Fott et Novákova (MACC 361) and Chlorella sp. (MACC 458) grown in a 14:10 light:dark (L:D) photoperiod. In 24 h experiments, cell division occurred during the dark period, and cells increased in size during the light period. Cytokinin profiles were similar in both strains, consisting of five cis-zeatin (cZ) and three N6-(2-isopentenyl)adenine (iP) derivatives. Cytokinin concentrations were low during the dark period and increased during the light period. In 48 h experiments using synchronized C. minutissima (MACC 361), half the cultures were maintained in continuous dark conditions for the second photoperiod. Cell division occurred during both dark periods, and cells increased in size during the light periods. Cultures kept in continuous dark did not increase in size following cell division. DNA analysis confirmed these results, with cultures grown in light having increased DNA concentrations prior to cell division, while cultures maintained in continuous dark had less DNA. Cytokinins (cZ and iP derivatives) were detected in all samples with concentrations increasing over the first 24 h. This increase was followed by a large increase, especially during the second light period where cytokinin concentrations increased 4-fold. Cytokinin concentrations did not increase in cultures maintained in continuous dark conditions. In vivo deuterium-labeling technology was used to measure cytokinin biosynthetic rates during the dark and light periods in C. minutissima with highest biosynthetic rates measured during the light period. These results show that there is a relationship between light, cell division, and cytokinins.