At gastrulation the primary mesenchyme cells of sea urchin embryos lose contact with the extracellular hyaline layer and with neighboring blastomeres as they pass through the basal lamina and enter the blastocoel. This delamination process was examined using a cell-binding assay to follow changes in affinities between mesenchyme cells and their three substrates: hyalin, early gastrula cells, and basal lamina. Sixteen-cell-stage micromeres (the precursors of primary mesenchyme cells), and mesenchyme cells obtained from mesenchyme-blastula-stage embryos were used in conjunction with micromeres raised in culture to intermediate ages. The micromeres exhibited an affinity for hyalin, but the affinity was lost at the time of mesenchyme ingression in vivo. Similarly, micromeres had an affinity for monolayers of gastrula cells but the older mesenchyme cells lost much of their cell-to-cell affinity. Presumptive ectoderm and endoderm cells tested against the gastrula monolayers showed no decrease in binding over the same time interval. When micromeres and primary mesenchyme cells were tested against basal lamina preparations, there was an increase in affinity that was associated with developmental time. Presumptive ectoderm and endoderm cells showed no change in affinity over the same interval. Binding measurements using isolated basal laminar components identified fibronectin as one molecule for which the wandering primary mesenchyme cells acquired a specific affinity. The data indicate that as the presumptive mesenchyme cells leave the vegetal plate of the embryo they lose affinities for hyalin and for neighboring cells, and gain an affinity for fibronectin associated with the basal lamina and extracellular matrix that lines the blastocoel.