Hypoxia is the alleged stimulus for initiation of increase of carbonic anhydrase II (CAII) and 2,3-diphosphoglycerate (2,3-DPG) synthesis of red blood cells from late chick embryos. The PO2-dependent regulation of red cell metabolism is mediated by unknown humoral factors [Million et al., Am. J. Physiol. 261 (Regulatory Integrative Comp. Physiol. 30): R1188-R1196, 1991]. In the present investigation we have analyzed whether interindividual differences in egg size (which result in different surface area-to-mass ratios) affect the timing of initiation of 2,3-DPG and CAII synthesis in late chick embryos. We also investigated the effect of extracellular adenine nucleotides on red cell organic phosphate pattern and O2 affinity to test whether the inhibitory effect of normal or elevated PO2 on 2,3-DPG synthesis and the concomitant increase of ATP (and O2 half-saturation pressure) can be mimicked by these agents. The results show that differences in egg size affect the timing of CAII and 2,3-DPG synthesis, indicating that PO2-dependent regulation of red cell function allows adjustment to the properties of the individual egg. We also found that extracellular ATP, which is rapidly degraded to AMP by red cell ectoenzymes, can alter the red cell phosphate pattern and O2 affinity, i.e., significantly increase red cell ATP, decrease red cell 2,3-DPG and O2 affinity, and thus mimic the effect of normoxia and hyperoxia. These findings suggest that extracellular adenine nucleotides may be involved in the PO2-dependent regulation of embryonic red cell metabolism.