Human cytochrome P450 3A4 forms a series of minor testosterone hydroxylation products in addition to 6b-hy-droxytestosterone, the major product. One of these, formed at the next highest rate after the 6b-and 2b-hydroxy prod-ucts, was identified as 1b-hydroxytestosterone. This product was characterized from a mixture of testosterone oxidation products using an HPLC-solid phase extraction-cryoprobe NMR/time-of-flight mass spectrometry system, with an estimated total of 6lgof this spectrometry established the formula as C19H29O3(MH + )