The existence of more than one chitin synthetase in fungal cells poses the question of whether these enzymes have similar or different localization. The subcellular distribution of chitin synthetases 1 and 2 (Chs1 and Chs2) was determined in cell-free extracts of Saccharomyces cerevisiae fractionated by sucrose density gradient sedimentation. Chs1 was examined in two strains: ATCC 26109, a wild-type strain, and D3C (MAT alpha ura3-52). Chs2 was investigated in a strain (D3B) freed of Chs1 by gene disruption (MATa his4 ura3-52 chs1::URA3). A prolonged, strong centrifugation (20 h at 265000 g) was necessary to cleanly resolve two major populations of chitin synthetase particles: chitosomes (a population of microvesicles of low buoyant density, d = 1.15 g ml-1) and plasma membrane (a population of vesicles of high buoyant density, d = 1.21 g ml-1). Chs1 and Chs2 were both present in chitosomes and plasma membrane, but the relative distribution of each chitin synthetase in these two membranous populations varied. Chs2 was much less abundant than Chs1 and required Co2+ rather than Mg2+ as a cofactor. A salient finding was the high sensitivity of chitosomal Chs2 to high centrifugal forces. The subcellular distribution of 1,3-beta-glucan synthetase was the same in the three strains studied, i.e. unaffected by the presence or absence of Chs1. Culture conditions affected the profiles of chitin and glucan synthetases: the relative abundance of Chs1 in chitosomes or plasma membrane was quite different in cells grown on two different media but the buoyant density was not affected; in contrast, there was shift in the buoyant density of the two peaks of 1,3-beta-glucan synthetase. We concluded that the subcellular localization of Chs1 and Chs2 remains the same despite genetic and other differences in the properties of these enzymes. We confirmed that 1,3-beta-glucan synthetase and chitin synthetase exhibit a partially different subcellular distribution-an indication that these two enzymes are mobilized through different secretory pathways.