In cell lines stably (KiKi) or reversibly (Ts) transformed by the k-ras oncogene originated from a differentiated rat throid line (FRTL5 cells), k-ras-induced transformation has been associated with an increased phospholipase A₂ activity. Here we provide evidence that this enzymic activity is phosphoinositide specific and leads to the formation of lysophosphatidylinositol. The levels of this lysolipid increased by 2–3-fold in ras-transformed cells (KiKi cells and Ts cells at the permissive temperature of 33°C) as compared to differentiated cells (FRTL5) or to Ts cells maintained at 39°C, i.e. at the temperature where ras-p21, the product of the ras oncogene, is inactive. Since another lysoderivative, lysophosphatidic acid, has been shown to be a mitogen, we have tested whether lysophosphatidylinositol could have a similar activity on thyroid cells. Lysophosphatidylinositol (10–100 μM) induced a 5–10-fold increase in [³H]thymidine incorporation in both FRTL5 and KiKi cells, whereas lysophosphatidic acid was active only in differentiated cells. Lysophosphatidylinositol (∼25 μM) and lysophosphatidic acid (50–100 μM) acted synergistically with insulin in increasing [³H]thymidine incorporation. Moreover, lysophosphatidylinositol at concentrations three-fold higher than those found to be mitogenic, inhibited the activity of the GTPase-activating protein. We conclude that lysophosphatidylinositol is a mitogen that might play a role in the modulation of k-ras transformed cell proliferation.