Many vertebrate species express two enzymes that are capable of catalysing the reduction of various isomers of biliverdin. Biliverdin-IX alpha reductase (BVR-A) is most active with its physiological substrate biliverdin-IX alpha, but can also reduce the three other biliverdin isomers IX beta, IX delta and IX gamma. Biliverdin-IX beta reductase (BVR-B) catalyses the reduction of only the IX beta, IX delta and IX gamma isomers of biliverdin. Therefore, the activity of BVR-A can be measured using biliverdin-IX alpha as a specific substrate. We now show that the dimethyl esters of biliverdin-IX beta and biliverdin-IX delta are substrates for BVR-B, but not for BVR-A. This provides a useful method for specifically assaying the activity of both BVR-A and BVR-B in crude mixtures, using biliverdin-IX alpha for BVR-A and the dimethyl ester of either biliverdin-IX beta or biliverdin-IX delta for BVR-B. Human BVR-A has been suggested as a pharmacological target for neonatal jaundice. Because of the absence of a crystal structure with biliverdin bound to BVR-A, we have investigated indirect ways of examining tetrapyrrole binding. In the present study, we report that a number of sterically locked conformers of 18-ethylbiliverdin-IX alpha are substrates for human BVR-A, and discuss the implications for the biliverdin binding site. The oxidation of bilirubin-IX alpha ditaurate to biliverdin-IX alpha ditaurate is also described. We show that biliverdin-IX alpha ditaurate is a substrate for human BVR-A and discuss the possibility of using a competing substrate, which is reduced to a water soluble and excretable rubin, as a prototypic inhibitor of BVR-A.