Both hypoxic preconditioning (HP) and chronic hypoxia (CH) protect the heart against injury caused by acute oxygen deprivation. The aim of the study was to find out whether the protection was preserved in ventricular myocytes isolated after HP and CH of intact rats. Animals were exposed to normobaric hypoxia (10% O2) for 8, 24 h or 21 days followed by 24 h of normoxia. Myocytes were isolated from the left (LVM) and right (RVM) ventricles and the septum (SEPM), and subjected to 25 min of “anoxia” (metabolic inhibition) followed by 30 min of reoxygenation (A/R). Viability and LDH release were evaluated at the beginning of the experiment, after anoxia, and after reoxygenation. Neither HP nor CH affected the viability of the myocytes per se. Initial LDH (total and released) did not differ in LVM and SEPM from any hypoxic group; in contrast, CH doubled LDH in RVM. Both HP and CH increased the viability after A/R of LVM and SEPM more than twice compared to normoxic controls (HP: 80% and 83%, CH: 70% and 79%, controls: 33% and 39%, respectively). The RVM of the CH group exhibited a somewhat smaller protection (59% vs. 32% in the controls), possibly reflecting a negative influence of hypertrophy. Similar protective effects were observed when LDH release was measured just after anoxia. LDH release after A/R was significantly lower in HP groups compared to normoxic controls while protection afforded by CH was less pronounced (HP: 109%, 107% and 114%, CH: 117%, 125% and 125%, controls: 132%, 129% and 139% in LVM, SEPM and RVM, respectively). In conclusion, the protective effects of both HP and CH were retained in subsequently isolated cardiomyocytes subjected to an acute A/R protocol. Although the increase in viability was comparable, protection against irreversible A/R injury (LDH release) was somewhat greater in myocytes isolated from HP rats compared to CH animals.