The in vitro biosynthesis of indole-3-butyric acid (IBA) was investigated in maize (Zea mays L.). Incubation of microsomal membranes from shoots and roots of dark-grown maize with labelled indole-3-acetic acid (IAA), acetyl coenzyme A (acetyl CoA) and ATP resulted in the formation of an unknown labelled reaction product, product X. Other coenzymes had very little effect on the formation of this product. No IBA was detected in this system. The labelled product was purified and fed to other cell fractions of dark-grown maize. Incubation with the organelle fraction using NADPH as a cofactor resulted in the conversion of product X to a new compound which had the retention time of IBA in HPLC. Attempts to identify product X led to the hypothesis that it is a conjugate of IAA with ADP. Microsomal membranes from light-grown shoots and roots converted IAA to IBA, as demonstrated by HPLC analysis. The identity of the IBA peak was confirmed by GC-mass spectrometry. Microsomal membranes from light-grown roots formed both IBA and little of product X. For the direct in vitro biosynthesis of IBA acetyl CoA was needed as a cofactor, and the reaction as enhanced by the addition of ATP or NADPH. The optimum pH of the in vitro formation of IBA was 6-7 in the roots and 5 in the shoots. Product X was formed only at neutral pH (7-8). The K_m value for IAA in the synthesis of IBA was 16 μm.