Carbon magnetic resonance T(1) relaxation and chemical shift measurements at 22.63 MHz establish hydrophobic aggregation of prostaglandin F(2alpha) in phosphate buffer solutions between 0.05 and 0.2 M. Analysis of the proton magnetic resonance spectra of prostaglandin F(2alpha) at 270 MHz by double resonance techniques yield all the proton-proton coupling constants for the five-membered ring indicating a favored half-chair conformation for the ring in which the dihedral angle for the C-8 and C-12 protons is close to 180 degrees . Effective correlation times derived from carbon magnetic resonance T(1) values for all the carbon atoms show segmental motion for ring carbon C-10 and in the aliphatic portions of both side chains, while the double bonded portions of the side chains and the ring carbons act as a more rigidly interconnected network. Chemical shift changes in the carbon magnetic resonance and proton magnetic resonance spectra upon aggregation suggest that the 5-6 double bond, C-7, and C-9 participate in the aggregation process.