The inositol phosphate hydrolyzing activity of human phospholipase Cδ1 (PLCδ1) is markedly inhibited when the enzyme is coexpressed with the human heart G_h/transglutaminase (TG) in human embryonic kidney cells. Because the cotransfection does not affect the amount of PLCδ1 in the cells, the depression of phospholipase activity probably is a result of a direct interaction between the two proteins. An ELISA procedure was employed to document the associations of purified TG preparations from a variety of tissues (human red cells, rabbit lens, guinea pig liver) with PLCδ1. Nucleotides (GTP > GDP > ATP > GMP = ADP, in order of decreasing efficiency) interfered with the formation of the PLCδ1:TG complex. A conformational change in the TG partner, occurring with nucleotide binding, is thought to be responsible for dissociating the two proteins. The structural rearrangement produces a remarkable shift in the anodic mobility of TG in electrophoresis: TG_slow + GTP ⇆ [TG:GTP]_fast. Altogether, our findings indicate that GTP controls PLCδ1 activity by releasing this protein from an inhibitory association with G_h/transglutaminase.