Experiments are presented to determine whether Rana pipiens gastrula cells differentiate in vitro as they do in vivo with respect to rate, morphology and spreading characteristics. Comparisons are made between presumptive germ layer cells isolated at late blastula, cultured for 24 h, and the same cell types form late gastrula cultured for 1 h (LeBlanc and Brick, 1981a). These cells are chronologically similar and should be morphologically similar at the end of their respective culture periods if in vivo and in vitro differentiation are proceeding in the same direction and rate. Five hour cultures of the same cell types were compared to 1 h cultures in media with enhanced Ca2+ (LeBlanc and Brick, 1981b) to study the role of Ca2+ as a modulator of spreading and production of cell protrusions, and the rate at which these occur, as these relate to morphogenetic movements. The data and inferences can be summarized as follows: (1) Some Rana pipiens late blastula and gastrula cells appear to differentiate morphologically in vitro similarly to their differentiation in vivo. (2) In vitro differentiation is autonomous indicating that determination occurred prior to late blastula, mid-gastrula or late gastrula depending on when cells were isolated. (3) The presence of two sub-populations is some germ layer cell isolate indicates determinative interactions had occurred prior to isolation. (4) Morphologic similarity of cells in standard culture for 5 h to the same cells in culture with additional Ca2+ for 1 h suggests that Ca2+ modulates spreading and projection formation and the rate at which these occur. (5) The upper cell surface of these cells may provide adhesive sites for plasmalemma projections from adjacent cells.