Due to a pH-sensitive effect in many fish hemoglobins (Hb), analytical errors may occur when mammalian Hb is used as a standard in quantitative spectrophotometric multicomponent analysis of fish blood. The aim of this work was to examine differences in the optical spectra of mammalian (human) and fish (farmed Atlantic cod) Hb subjected to pH 7.4 and 6.5. The absorption spectra of the common derivatives, deoxy- (HHb), oxy- (OHb), carboxy- (COHb), and methemoglobin (metHb), were determined in the spectral range of 450-700 nm. The metHb spectra of fish differed considerably from the corresponding human Hb spectra, whereas only minor differences in OHb, HHb, and COHb were found. Cod Hb was significantly (P < 0.05) influenced by a drop in pH compared to mammalian Hb. This resulted in deoxygenation of the Hb and increased autoxidation. For human Hb, a pH-independent isosbestic point in the spectra of OHb, HHb, and metHb at 523 nm was found. This isosbestic point was not found in the absorption spectra of cod Hb. In conclusion, spectra of cod metHb and human metHb behave differently. This must thus be taken into account in spectrophotometric multicomponent analysis. Ideally, Hb in muscle or blood should be determined by comparison to a standard made from the same species.