Fumonisins are mycotoxins produced by the maize pathogen Gibberella fujikuroi mating population A and frequently contaminate maize. Wild-type G. fujikuroi produces four B-series fumonisins, FB1, FB2, FR3 and FB4. These toxins are identical in structure except for the number and positions of hydroxyls along their linear carbon backbone. To elucidate the genetic and biosynthetic relationships among these fumonisins, we conducted meiotic and biochemical analyses of G. fujikuroi mutants with altered fumonisin production that resulted from defective alleles at three loci, Fum1, Fum2 and Fum3. These mutants produced either no fumonisins, only FR2 and FB4, or only FR3 and FR4. Genetic analyses revealed the orientation of the Fum loci along linkage group 1 of the fungus. The mutants were grown together in pair-wise combinations to determine if their fumonisin production phenotypes could be complemented. When FR3- and FB2-producing mutants were grown together, complementation occurred. However, when a nonproducing mutant was grown with a FR2- or FB3-producing mutant, complementation did not occur or was incomplete. When purified FR2, FR3, or FB4 was fed to mutant cultures, FR4 was converted primarily to FR2, FR3 was converted to FB1 and FB2 was not converted. The results from these assays suggest a previously unrecognized branch in the fumonisin biosynthetic pathway.