Calcium is shown to have several roles in the adhesion of embryonic sea urchin cells. Using an assay that subdivides a cell interaction into sequential steps it is shown that Ca2+ has distinct roles in at least two separate steps. The initial binding step between blastomeres is Ca2+-sensitive, and is shown to require divalent cations themselves rather than replacement of a macromolecular material that might be removed in the absence of Ca2+. The secondary steps of adhesion involve a strengthening of the initial contacts. Ca2+ is shown to be required for at least one and probably more than one of these steps. In addition, the extracellular matrix molecule called hyalin is known to be sensitive to Ca2+ for its polymerization. Hyalin may be involved in the initial steps of cell binding measured by the assay in that cell-cell binding is partially blocked by a Fab antibody fragment directed against hyalin. Finally, two extracts, known from other studies to enhance cell aggregation, were examined for their possible relationship to the Ca2+-sensitive binding of cells. A butanol extract was shown to enhance initial binding in a Ca2+-independent manner. Cells extracted with butanol were greatly reduced in adhesiveness even in the presence of Ca2+. The second extract was obtained from cells by treatment of embryos with divalent cation-free saline. This Ca2+-sensitive factor is shown to contain hyalin which may account for some of its observed activity, though not necessarily all of it. Divalent cation-free-extracted cells regain full adhesiveness immediately upon restoration of Ca2+. Thus the extract is not essential for cell adhesiveness. Together these data show that divalent cations have several roles in the multistepped process called adhesion.