Changes in the cellular mass and the cellular concentration of the highly phosphorylated inositol derivatives inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P5) and inositol hexakisphosphate (InsP6) were observed during a complete cell cycle of proliferating rat thymocytes. Inositol polyphosphates were determined by a recently developed anion-exchange HPLC/post-column complexometric dye system (Mayr, G. W. (1988) Biochem. J. 254, 585-591), and data were expressed as mass amounts per cell number or as absolute intracellular concentrations based on cell volume determinations by the cell analyzer system CASY 1. After a very early transient rise in both Ins(1,3,4,5,6)P5 and InsP6 at the beginning of the cell cycle, a decrease of the intracellular concentration of both compounds occurred which then remained on a low level between 24 and 48 h. Between 48 and 72 h the cells divided resulting in a decrease of the cell volume by a factor of approximately 2. During this phase a pronounced increase of Ins(1,3,4,5,6)P5 and InsP6 was observed. These increases were already seen when the data were expressed as mass per cell number, but were even enhanced when expressed as absolute intracellular concentrations. In conclusion, we present as a novel finding long term changes in the intracellular concentrations of Ins(1,3,4,5,6)P5 and InsP6 in proliferating thymocytes. This may indicate a role for these compounds during cell cycle progression.