Abscisic acid (ABA) is an important plant hormone involved in controlling normal growth and development in higher plants. The fungus Cercospora rosicola synthesizes large quantities of ABA (Assante et al., 1977), which has made it a useful organism for studies of ABA biosynthesis;The purpose of this study is to examine the ABA biosynthetic pathway in C. rosicola. Feeding experiments with labeled mevalonic acid (MVA), farnesyl pyrophosphate (FPP), and 1[superscript]'-deoxy-ABA have confirmed the conversion of these intermediates into ABA. This conversion was inhibited in the fungicide triarimol-treated cultures fed with MVA and FPP, but not 1[superscript]'-deoxy-ABA;The development of a cell-free enzyme system from fungal extracts capable of converting any of these intermediates into ABA has not been previously reported. A cell-free enzyme system has been developed in our laboratory to convert 1[superscript]'-deoxy-[superscript]2H-ABA into [superscript]2H-ABA. The reaction products were chromatographed by reverse phase HPLC. The presumptive ABA fractions were collected and [superscript]2H-ABA was quantified by GC-MS using a [superscript]2H-ABA standard curve. 1[superscript]'-deoxy-[superscript]2H-ABA was converted to an average of 1.47 pmol [superscript]2H-ABA mg[superscript]-1 protein min[superscript]-1. Most of the enzymatic activity was found in the microsomal fraction. The reaction required NADPH and was enhanced by FAD; it was not inhibited by triarimol. These data, taken together, suggest that cytochrome P-450 may be involved in some oxidative reactions prior to 1[superscript]'-deoxy-ABA in the ABA biosynthetic pathway;The phytopathogenic fungi Gnomonia veneta and Cercospora mori infect sycamore and mulberry leaves, respectively. These fungi do not appear to produce ABA in liquid cultures. However, infected leaves with these fungi contain elevated levels of ABA and abscise prematurely.