Three criteria have been used to distinguish among different systems of embryonic cell adhesion: dependence on Ca2+, involvement of particular cell-surface molecules, and binding specificity. The characterization of the adhesion with respect to cell-surface molecules was carried out by using specific antibodies against the neural and liver cell adhesion molecules (N-CAM and L-CAM) and antibodies raised against retinal cells prepared by limited trypsinization in the presence of Ca2+ (called "T/Ca cells"). Aggregation of cells prepared from retina or brain without Ca2+ did not require Ca2+ and was inhibited by anti-(N-CAM) antibodies but not by anti-(L-CAM) or anti-T/Ca cell antibodies. In contrast, cells obtained from the same tissues in the presence of Ca2+ did require Ca2+ to aggregate. This aggregation was inhibited by anti-T/Ca cell antibodies but not by anti-(N-CAM) or anti-(L-CAM) antibodies. Hepatocyte aggregation also required Ca2+ and was inhibited only by anti-(L-CAM) antibodies. These results define three antigenically distinct cell adhesion systems in the embryo and raise the possibility that additional systems will be found. The neural Ca2+-independent system displayed a limited tissue specificity, mediating binding to neural but not liver cells. In contrast, the Ca2+-dependent systems of both neural and liver cells caused binding to all cell types tested. The Ca2+-dependent system was most active in retinal cells from 6-7 day embryos, whereas the Ca2+-independent system was most active at later times during development. In addition, treatments that inhibited the Ca2+-independent or Ca2+-dependent systems had very different effects on the fasciculation of neurites from dorsal root ganglia. All of the results suggest that Ca2+-independent and Ca2+-dependent adhesion systems play different functional roles during embryogenesis.