[3H]Inositol and [32P]Pi labelling of the aquatic plant Spirodela polyrhiza L. revealed the presence of PtdIns(3,4)P2, in addition to PtdIns3P, PtdIns4P and PtdIns(4,5)P2 previously identified [Brearley and Hanke (1992) Biochem. J. 283, 255-260]. PtdIns(3,4,5)P3 was not detected. Throughout a 40 min [32P]Pi-labelling period the specific radioactivity of the gamma-phosphate of ATP and of the ATP pool as a whole increased. Chemical and enzymic dissection of phosphoinositides obtained from plants labelled for 35 min with [32P]Pi showed that over 99.7% of the label in PtdIns3P and PtdIns4P was accounted for by the monoester phosphates. The 3- and 4-monoester phosphates of PtdIns(3,4)P2 accounted for 23.1% and 76.6% respectively of the label, whereas the 4- and 5-monoester phosphates of PtdIns(4,5)P2 accounted for 21.1% and 78.6% respectively. These results are consistent with the synthesis of PtdIns(4,5)P2 via PtdIns4P. The labelling of the individual phosphates of PtdIns(3,4)P2 is, however, inconsistent with synthesis from PtdIns(4,5)P2 via PtdIns(3,4,5)P3, but instead suggests that PtdIns(3,4)P2 is synthesized by 4-phosphorylation of PtdIns3P. These results afford the first evidence that in plants in vivo, synthesis of PtdIns(4,5)P2 follows the pathway described in animal cells and also that plants possess PtdIns3P 4-kinase activity similar to that reported from animal cells.