Thrombospondin-1 is an adhesive glycoprotein that is involved in cellular attachment, spreading, migration, and proliferation. To date, four genes have been identified that encode for the members of the thrombospondin gene family. These four genes are homologous to each other in the EGF-like (type 2) repeats, the calcium-binding (type 3) motifs, and the COOH-terminal. The latter has been reported to be a cell-binding domain in thrombospondin-1. Phylogenetic trees have been constructed from the multisequence alignment of thrombospondin sequences from human, mouse, chicken, and frog. Two different algorithms generate comparable results in terms of the topology and the branch lengths. The analysis indicates that an early form of the thrombospondin gene duplicated about 925 million years ago. The gene duplication that produced the thrombospondin-1 and -2 branches of the family is predicted to have occurred 583 million years ago, whereas the gene duplication that produced the thrombospondin-3 and -4 branches of the family is predicted to have occurred 644 million years ago. These results indicate that the members of the thrombospondin gene family have existed throughout the evolution of the animal kingdom and thus probably participate in functions that are common to most of its members.