Lysophosphatidylinositol (LPI) is an endogenous ligand for GPR55, a putative novel type of cannabinoid receptor. In this study, we first examined the effects of LPI on p38 mitogen-activated protein kinase in HEK293 cells expressing GPR55. LPI induced the rapid phosphorylation of p38 mitogen-activated protein kinase in GPR55-expressing cells. No apparent effect was observed in the vector-transfected cells. The exposure of GPR55-expressing cells to LPI also triggered the phosphorylation of activating transcription factor 2 downstream of the p38 mitogen-activated protein kinase. Treatment of the cells with Y-27632 [a Rho-associated kinase (ROCK) inhibitor] blocked the LPI-induced phosphorylation of p38 mitogen-activated protein kinase and activating transcription factor 2, suggesting that the Rho-ROCK pathway is involved in these cellular responses. Notably, GPR55 was found to be abundantly expressed in lymphoid organs such as the spleen and thymus. We obtained evidence that rapid phosphorylation of p38 mitogen-activated protein kinase and activating transcription factor 2 also takes place in IM-9 lymphoblastoid cells, which naturally express GPR55, after stimulation with LPI. These results suggest that GPR55 and its endogenous ligand LPI play essential roles in the homoeostatic responses to stress signals in several mammalian tissues and cells including certain types of immune cells.