Evaluation of apoptotic processes downstream of the mitochondrion reveals caspase-9- and low levels of caspase-3-like activities in partly purified extracts of A. franciscana embryos. However, in contrast to experiments with extracts of human hepatoma cells (C3A), cyt-c fails to activate caspase-3 or -9 in extracts from A. franciscana. Furthermore, caspase-9 activity is sensitive to exogenous calcium. Addition of 5 mM calcium leads to a 4.86 ± 0.19 fold (n = 3; ± SD) increase in activity, which is fully prevented with 150 mM KCl. As with mammalian systems, high ATP (>1.25 mM) suppresses caspase activity in A. franciscana extracts. A strong inhibition of caspase-9 activity was also found by GTP. Comparison of GTP-induced inhibition of caspase-9 at 0 and 2.5 mM MgCl₂ indicates that free (non-chelated) GTP is likely to be the inhibitory form. The strongest inhibition among all nucleotides tested was with ADP. Inhibition by ADP in the presence of Mg²⁺ is 60-fold greater in diapause embryos than in post-diapause embryos. Because ADP does not change appreciably in concentration between the two physiological states, it is likely that this differential sensitivity to Mg²⁺-ADP is important in avoiding caspase activation during diapause. Finally, mixtures of nucleotides that mimic physiological concentrations in post-diapause and diapause states underscore the depressive action of these regulators on caspase-9 during diapause. Our biochemical characterization of caspase-like activity in A. franciscana extracts reveals that multiple mechanisms are in place to reduce the probability of apoptosis under conditions of energy limitation in this embryo.