Apolipoprotein D (ApoD) constitutes an atypical lipoprotein in so far as it is predominantly found associated with HDL particles but belongs to the lipocalin structural family. Apart from its involvement in serum lipid transport it is abundant in various tissues, and differing physiological functions have been ascribed to it. We have now developed an E. coli expression system that permits the efficient production of biochemically homogeneous ApoD via secretion into the bacterial periplasm. Detailed ligand binding studies by fluorescence titration revealed that progesterone and arachidonic acid are complexed with dissociation constants both in the 1 microM range, whereas the presumed ligands pregnenolone, bilirubin and E-3M2H are not recognized by the recombinant protein. In contrast with previous reports it thus appears that ApoD discriminates well in its binding function between closely related compounds.