We investigated the molecular mechanisms regulating differences in mitochondrial volume density between heart ventricles of Antarctic notothenioids that vary in the expression of hemoglobin (Hb) and myoglobin (Mb). In mammals, peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) and nuclear respiratory factor 1 (NRF-1) stimulate mitochondrial biogenesis and maintain mitochondrial density in muscle tissues. We hypothesized that these factors would also maintain mitochondrial density in the hearts of Antarctic notothenioids. The percent cell volume occupied by mitochondria is significantly lower in hearts of the red-blooded notothenioid Notothenia coriiceps (18.18+/-0.69%) in comparison with those of the icefish Chaenocephalus aceratus (36.53+/-2.07%), which lacks both Hb and cardiac Mb. Mitochondrial densities are not different between hearts of N. coriiceps and Chionodraco rastrospinosus, which lacks Hb, but whose heart expresses Mb. Despite differences in mitochondrial volume density between hearts of N. coriiceps and C. aceratus, the levels of transcripts of the genes encoding PGC-1alpha, NRF-1 and citrate synthase, and the copy number of mitochondrial DNA do not differ. Our results indicate that the high mitochondrial densities in hearts of C. aceratus may result from an increase in organelle size. The surface-to-volume ratio of mitochondria from N. coriiceps is 1.9-fold greater than that of mitochondria from C. aceratus. In addition, the levels of PGC-1alpha correlate with mitochondrial density in muscle tissues of notothenioids possessing mitochondria of similar size and morphology. Finally, the levels of PGC-1alpha are 4.6-fold higher in the aerobic pectoral adductor muscle in comparison with the glycolytic skeletal muscle of N. coriiceps. The potential physiological significance of an increase in mitochondrial size in hearts of Antarctic icefishes is discussed.