1. 1. A non-hydrolyzable synthetic phosphatidic acid analogue, 2-hexadecoxy-3-octadecoxypropylphosphonic acid, shows considerable activity as an inhibitor of pig kidney phosphatidate phosphohydrolase ( l-α-phosphatidate phosphohydrolase, EC 3.1.3.4). Under specified conditions it can reduce the rate of hydrolysis of egg-lecithin phosphatidate by 96% in equimolar substrate-inhibitor concentrations. Maximal inhibition requires a period of preincubation of the analogue with enzyme; most of this effect occurs within the first few minutes and reaches an almost constant maximal inhibition at 30 min. 2. 2. Kinetic data of the inhibition are difficult to interpret definitively, but reactivation studies indicate an appreciable degree of irreversibility in the action of the inhibitor. 3. 3. Comparison with other potential inhibitory compounds suggests that 2-hexadecoxy-3-octadecoxypropylphosphonate does not exert its effect by surface activity but that its structure is at least partially specific for the inhibition. 4. 4. The inhibition depends in part on the degree of dispersion of the analogue. The hydrolysis of saturated phosphatidic acids is also inhibited by the analogue under conditions similar to the inhibition of enzymic hydrolysis of egg phosphatidic acid. Bacterial growth studies suggest that 2-hexadecoxy-3-octadecoxypropylphosphonate is not a general enzyme inhibitor.