The essential fungal cell-wall polymer (1,3)beta-glucan is synthesized by the enzyme (1,3)beta-glucan synthase. This enzyme, which is the target of the echinocandin and pneumocandin families of fungicidal antibiotics, is a complex composed of at least two proteins, Rho1p and Fks1p. Homologs of the yeast FKS1 gene have been discovered in numerous fungi, and existing evidence points to, but has not yet proved, Fks1p being the catalytic subunit of (1,3)beta-glucan synthase. We have purified (1,3)beta-glucan synthase from Neurospora crassa approximately 400-fold enrichment and labeled the substrate-binding protein by using a UDP-glucose analog, 5-azido-[beta-(32)P]-UDP-glucose. UDP-glucose-binding proteins were photo-crosslinked to the substrate analog and identified from SDS-PAGE gels by Quadrupole time-of-flight mass spectrometry by sequencing the tryptic peptides. Two plasma membrane proteins were labeled FKS and H(+)-ATPase. These results suggest that FKS appears to be the substrate-binding subunit of (1,3)beta-glucan synthase.