Groups of inside cells (ICs) and inner cell masses (ICMs) were isolated from individual mouse embryos between the late morula and day expanded blastocyst stages using a modified immunosurgical procedure, and their purity and developmental potential were assessed in vitro. Several different techniques failed to detect the presence of viable contaminating outside cells on ICs isolated from any of the stages studied. The numbers of inside cells isolated from the earlier stages, counted in air-dried preparations, were considerably higher than previous estimates from serial sections; whereas the numbers isolated from expanded blastocysts were in reasonable agreement. Thus the proportion of inside cells recovered by immunosurgery decreases over this period of development. In view of the evidence that inside cells divide at a faster rate than outside cells at these stages, it is argued that there may be an outward movement of inside cells capable of forming trophectoderm, during expansion of the blastocyst. ICs and ICMs in vitro were observed to develop in one of two distinct ways according to the stage at which they were isolated. ICs from late morulae and some early cavitating blastocysts formed blastocyst-like vesicles over a period of 24-36 h in culture. The presence of trophectoderm cells in these vesicles was confirmed by the persistence of giant cells after ectopic transfer. In contrast ICs from a minority of early cavitating blastocysts, and all ICMs from day expanded blastocysts did not form vesicles, but proliferated endodermlike cells. Thus at least some inside cells do not appear to lose the capacity to form trophectoderm and do not become committed to an ICM fate until after the initial formation of the blastocoel cavity.