Streptococcus gordonii glucosyltransferase polymerizes sucrose to form glucans, which confer a hard, sucrose-promoted phenotype (Spp+) to colonies on sucrose agar plates. The glucosyltransferase structural gene, gtfG, is positively regulated by the upstream determinant, rgg. Strain Challis undergoes a spontaneous, reversible phase variation between high (Spp+) and low (Spp-) levels of glucosyltransferase activity. Representative strains were examined to gain insights into the basis of glucosyltransferase phase variation. Western blots indicated that the level of glucosyltransferase activity was related to the amount of extracellular glucosyltransferase protein produced by Spp- and Spp+ strains. The nucleotide sequence of rgg and gtfG of the Spp- strain CH97 was found to be identical to that of the Spp+ parent, indicating that DNA differences in these regions are not the basis for glucosyltransferase phase variation. Indeed, 13C-NMR spectroscopy suggested that glucans synthesized by strain CH97 glucosyltransferase were similar to those synthesized by glucosyltransferase of the Spp+ parental strain, indicating a quantitative rather than qualitative change. However, one Spp- strain, CH1C1, had a point mutation in rgg; replacement of the parent rgg with the CH1C1 allele resulted in decreased levels of glucosyltransferase protein and activity. The results indicate that glucosyltransferase phase variation can occur in more than one way, and suggest that glucosyltransferase regulation may involve distally located regulatory gene(s) that affect rgg and/or gtfG expression.