Molecular and biochemical studies investigating the biosynthesis of gamma-linolenic acid (GLA), a nutritionally important polyunsaturated fatty acid, were implemented in Mucor rouxii by supplementation with exogenous fatty acids. Compared to the fatty acid-free cultures, addition of C18:2Δ9,12 to the cultures showed up to threefold decrease in the GLA content from the beginning of the culture, whereas a significantly increased GLA amount (P≤0.05) was found after 9 h of growth in thelogarithmic cultures fed with stearic acid or oleic acid. Time-course studies of gene expression showed concomitant changes in the messenger RNA (mRNA) levels of the M. rouxii Δ6-, Δ9-, and Δ12-desaturases upon exposure to fatty acids, and these responses were rapid and transient. However, the expression profiles were different depending on the type of exogenous fatty acids supplementing the cultures. Up-regulation of the three desaturase transcripts was detected after addition of C18:0, whereas supplementation of unsaturated fatty acids led to a decrease in mRNA levels. This study showed that the three desaturase genes involved in fatty acid desaturation in M. rouxii are coregulated in response to exogenous fatty acids.