The unique Physarum lysophosphatidic acid, PHYLPA, having a cyclopropane in the fatty acid moiety and a cyclic phosphate at C-2 and C-3 positions of the glycerol, inhibited proliferation of human fibroblast cells, TIG-3 and TIG-7, which were cultured in a chemically defined (serum-free) medium. The cells at S- and M-phases proceeded to G2- and G1-phases, respectively, and most of cells were arrested at G1- or G2-phase during PHYLPA treatment. The growth was recovered when PHYLPA was removed from the medium. In the presence of serum, PHYLPA did not show obvious inhibitory effects, indicating the existence of a factor(s) which neutralizes the antiproliferative activity of PHYLPA. PHYLPA elicited an increase in 3',5'-cyclic adenosine monophosphate (cAMP) in a biphasic fashion in fibroblast cells. It also elicited inositol phosphate accumulation, as well as a transient rise in cytoplasmic free Ca2+ ion.