The echinocandins are a family of cyclic lipopeptides with potent antifungal activity. These compounds inhibit the synthesis of -1,3-glucan in fungi. The new semisynthetic echinocandin LY303366 was derivatized to produce a photoactivatable cross-linking echinocandin analog with antifungal activity. This analog was radioiodinated and used as a probe in microsomal membrane preparations of Candida albicans which contain glucan synthase activity. The photoaffinity probe identified two major proteins of 40 and 18 kDa in both membrane preparations. Labeling of these proteins was specific in that it required irradiation with UV light and was effectively competed against with unlabeled echinocandin analogs. In addition, the abilities of echinocandin analogs to compete with the photoaffinity probe correlated to their relative antifungal potencies and glucan synthase inhibition. The 40-kDa protein was isolated, and partial sequences were obtained from internal peptide fragments of the protein. Analysis of the sequences of these internal peptides of the 40-kDa protein revealed that it was a new protein not previously described as being involved in glucan synthesis or the mode of action of echinocandins.