When Chlamydomonas eugametos gametes were incubated in carrier-free [32P]P1, the label was rapidly incorporated into PtdInsP and PtdInsP2 and, after reaching a maximum within minutes, was chased out by recirculating unlabelled P1 in the cell. This pulse-chase labelling pattern reflects their rapid turnover. In contrast, 32P incorporation into the structural lipids was slow and continued for hours. Of the radioactivity in the PtdInsP spot, 15% was in PtdIns3P and the rest in PtdIns4P, and of that in the PtdInsP2 spot, 1% was in PtdIns(3,4)P2 and the rest in PtdIns(4,5)P2, confirming the findings by Irvine, Letcher, Stephens and Musgrave [(1992) Biochem. J. 281, 269-266]. When cells were labelled with carrier-free [32P]P1, both PtdInsP isomers incorporated label in a pulse-chase-type pattern, demonstrating for the first time in a plant or animal system that D-3 poly-phosphoinositides turn over rapidly in non-stimulated cells, with kinetics similar to those shown by the D-4 isomers. In animal systems such lipids are already established as signalling molecules, and the data suggest that a similar role must be sought for them in lower plants such as Chlamydomonas.