Monascus anka produces structurally related yellow and orange pigments. The biogenetic relationship between the pigments is not yet clear. It has been proposed that the yellow pigments(ankaflavin and monascin) are nonenzymatically formed from the orange pigments(monascorubrin and rubropunctatin). To elucidate which of the two colors appears earlier in the biogenetic sequence, cell-free biosynthesis of the pigments is attempted. A cell-free extract from Monascus anka was prepared with mycelial mat from standing culture and incubated with [1-^(14)C] acetate. The ethyl ether extract of the reaction mixture was chromatographed on TLC plate and radiochromatogram was obtained. The radiochromatogram clearly indicated formation of two products. One product with higher Rf value did not match with any pigment present in the mycelia. Position of another pigment with lower Rf value corresponded with orange pigments) in at least two solvent systems, thus establishing the earlier biosynthesis of the orange pigment(s). The conversion was promoted with addition of NADPH and inhibited with dithiothreitol which is a sulfhydryl protecting reagent. To enhance the conversion rate of acetate into the pigment(s), addition of sterol synthesis inhibitor was studied. Effect of the inhibitors (chlorocholine chloride and miconazole) was examined with the fungus in vivo. CCC showed more pronounced effect in shifting the metabolism of acetate toward production of the pigment,. The compound was added to the reaction mixture and found to be an effective modulator of the metabolism in vitro as well as in vivo. Implication of the result in the pigment biosynthesis and application in the pigment production will be discussed.