Membrane lipid fluidity was reexamined in red blood cells and ghosts from adults and newborns. Fluorescence anisotropies of the hydrophobic probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and perylene were significantly and substantially greater in fresh intact red cells from newborns than from adults; however, no significant difference was detected with the polar fluorophores, 12-(9-anthroyl) stearic acid and retinol. These results suggest that probes in the hydrophobic core of the membrane have less motional freedom in red cells from newborns than from adults, whereas probe motional freedom in the polar lipid headgroup regions of the membranes is similar for both cell types. DPH fluorescence anisotropy increased upon making white ghosts or upon storage of blood. Temperature studies indicated that DPH fluorescence anisotropy in fresh intact neonatal red cells is increased by an amount corresponding to that produced by cooling adult red cells by 22° C. Elevated intracellular calcium decreased red cell filterability without affecting DPH fluorescence anisotropy of ghost membranes. This result suggests that the effect of calcium in reducing filterability is independent of alterations in membrane lipid motional freedom. It is unlikely that the decreased lipid motional freedom of red cells from newborns contributes significantly to their decreased filterability. (Pediatr Res24: 433–437, 1988)