Two distinct murine monoclonal antibodies, designated MA-I and MA-II, and limited proteolysis with thrombin and trypsin have been used to probe the structure of human platelet thrombospondin. The results indicate that each of the constituent chains of thrombospondin comprise four distinct polypeptide segments. The production of these segments is influenced by the presence of calcium, the enzyme employed, the temperature of digestion, and the enzyme-to-substrate ratio. Thrombin digestion in the presence of calcium results in the release of a 30,000-dalton fragment, designated segment I, which contains the epitope for MA-II and the heparin-binding site. Prior EDTA treatment results in the concomitant cleavage of a 25,000-dalton fragment, designated segment IV, from the other terminus. Limited tryptic digestion in the absence of calcium produces a 47,000-dalton fragment (segment III) which is adjacent to segment IV. Segment III contains the epitope for MA-I. Segment II is an 85,000-dalton fragment which contains the interchain disulfide bonds. Calcium inhibits proteolysis at cleavage sites between segments II and III and between segments III and IV. In the presence of calcium, an 85,000-dalton fragment is produced, which is derived from portions of segments II, III, and possibly IV. Electron microscopy of platinum replicas produced by low angle rotary shadowing reveals that thrombospondin is composed of four well-defined globular regions connected by thin flexible regions. Three of the globular regions, designated globular region C, appear to be at the ends of the three thin connecting regions. The fourth globular region, designated globular region N, appears to be close to the site where the chains are cross-linked. Globular region N can be resolved into three separate smaller globular structures which are 70 +/- 7.1 A in diameter. This region is selectively removed by thrombin digestion in the presence of calcium and binds a monoclonal antibody directed against the heparin-binding peptides. These data indicate that globular region N comprises the three NH2-terminal portions (segment I) from each of the three chains of thrombospondin. Globular region C is located at the ends of each of the three thin connecting regions which are each approximately 291 +/- 46 A long. The removal of calcium results in a decrease in the size of globular region C from 118 +/- 18.6 A to 80 +/- 7.4 A and an increase in the length of the adjacent thin connecting region to 383 +/- 30 A.(ABSTRACT TRUNCATED AT 400 WORDS)