New inhibitor or inhibitors of nicotinamide-adenine dinucleotide-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) activity were found in breast milk but not in powdered milk. Milk samples were obtained manually from 23 puerperae who had delivered at 38 to 41 weeks. The inhibition pattern was competitive and noncompetitive. The percentage of inhibition of PGDH activity kept at 4.0 degrees C increased to a maximum at 12 hours after milk sampling and decreased thereafter. The percentage of inhibition of each milk sample decreased gradually with the duration postpartum. In addition, the inhibitor or inhibitors showed a smaller molecular weight than 1000 daltons and were partially resistant to heat and trypsin. Moreover, they were easily soluble in petroleum ether at various pH values, although the adsorption to C18 cartridge was inadequate. These new factor or factors may increase the benefits of breast feeding.