The thylakoid polypeptides of the cyanobacterium Anacystis nidulans R2 were analyzed by Triton X-114 phase fractionation [C. Bordier (1981) J. Biol. Chem. 256, 1604-1607, as adapted for photosynthetic membranes by T.M. Bricker and L.A. Sherman (1982) FEBS Lett. 149, 197-202]. In this procedure, polypeptides with extensive hydrophobic regions (i.e., intrinsic proteins) form mixed micelles with Triton X-114, and are separated from extrinsic proteins by temperature-mediated precipitation of the mixed Triton X-114-intrinsic protein micelles. The polypeptide pattern after phase fractionation was highly complementary, with 62 of the observed 110 polypeptide components partitioning into the Triton X-114-enriched fraction. Identified polypeptides fractionating into the Triton X-114 phase included the apoproteins for Photosystems I and II, cytochromes f and b6, and the herbicide-binding protein. Identified polypeptides fractioning into the Triton X-114-depleted (aqueous) phase included the large and small subunits of RuBp carboxylase, cytochromes c550 and c554, and ferredoxin. Enzymatic radioiodination of the photosynthetic membranes followed by Triton X-114 phase fractionation allowed direct identification of intrinsic polypeptide components which possess surface-exposed regions susceptible to radioiodination. The most prominent of these polypeptides was a 34-kDa component which was associated with photosystem II. This phase partitioning procedure has been particularly helpful in the clarification of the identity of the membrane-associated cytochromes, and of photosystem II components. When coupled with surface-probing techniques, this procedure is very useful in identifying intrinsic proteins which possess surface-exposed domains. Phase fractionation, in conjunction with the isolation of specific membrane components and complexes, has allowed the identification of many of the important intrinsic thylakoid membrane proteins of A. nidulans R2.