Novel paradigms for CYP24A1 inhibitor development are needed to circumvent existing efficacy and toxicity issues related to human therapeutics in this class. We hypothesize that improved structural knowledge of CYP24A1 in complex with natural substrates, inhibitors and/or its redox partner protein, adrenodoxin (Adx) is required to facilitate the next generation of CYP24A1 inhibitor design. To this end, we have developed truncated expression constructs for both rat CYP24A1 (Δ51) and bovine Adx (Δ108), which allow us to purify a stable and reversible state of the CYP24A1:Adx complex, for use in ongoing X-ray crystallographic studies. Spectral characterization of the reversible complex revealed that Adx binding enhanced the stability of the enzyme-substrate complex, despite lowering the ligand binding affinity of the free enzyme, for 1,25(OH) 2 D 2 , over 9-fold. Truncation of CYP24A1's flexible N-terminus (Δ51) improved the enzyme's ability to recruit substrate, without altering Adx's ability to stabilize the ligand-bound form. We also found that several common crystallization detergents, including CHAPS, inhibit ligand binding to the CYP24A1:Adx complex at concentrations well below their reported critical micelle concentration (CMC) values. Ultimately, this research provides a useful platform and framework for the study of conformationally complex, membrane–protein complexes, in the ligand-bound state.