Bilirubin was determined by direct spectrophotometry and by the candidate reference method of Doumas et al. (Clin. Chem. (1985) 31; 1789-1798) in a group of more than 230 newborns, and the results were compared. Direct spectrophotometry by Vink et al. (Clin. Chem. (1988) 34; 67-70) gives the following regression equation in comparison with the reference method: y = 0.967x + 6.57 with r = 0.9874. Comparison of direct spectrophotometry at 463 and 582 nm (for the simultaneous determination bilirubin and HbO2) with the reference method gave the following regression equation: y = 0.971x + 8.16 with r = 0.9814. Two methods of direct two-wavelength spectrophotometry showed statistically insignificant differences, using significance levels p = 0.05 and p = 0.01, in comparison with the reference method. In our group of newborns we determined serum HbO2 concentrations from 0.2 g/l (as a 0.025 fractile) to 2.6 g/l (as a 0.975 fractile) with a median value of 0.76 g/l. In a micromodification, the reference method is insensitive to HbO2 in concentrations of up to 5 g/l. Both direct spectrophotometric methods are insensitive to HbO2 concentrations up to 25 g/l. Direct spectrophotometric methods give results identical to those of the reference method and therefore are very suitable for use in neonatology.