Glucans synthesized by glucosyltransferase enzymes of oral streptococci facilitate bacterial accumulation on surfaces. The Streptococcus gordonii glucosyltransferase gene, gtfG, is positively regulated by rgg, which encodes a putative cytoplasmic protein. The gtfG promoter and ribosomal binding sequences are located within a DNA inverted repeat immediately downstream of rgg. Polycistronic rgg-gtfG as well as rgg- and gtfG-specific transcripts are associated with this chromosomal region. Previous studies have shown that the rgg product acts in trans near the gtfG promoter to increase the level of gtfG transcript, but it does not affect the level of rgg-gtfG transcript. To further analyze regulation by rgg, a series of strain Challis derivatives was constructed and glucosyltransferase activities were determined. Strains in which rgg was separated from gtfG by integrated vector sequences had decreased levels of glucosyltransferase activity; plasmid-borne rgg could not increase activity to parental levels. As expected, strains with chromosomal deletions involving the rgg structural gene and either the rgg or gtfG promoter also showed decreased glucosyltransferase activity. Plasmid-borne rgg could increase glucosyltransferase activity only in strains which had a 36-bp chromosomal region beginning 72 nucleotides upstream of the gtfG transcriptional start site. Results suggest that these nucleotides, located within the 3' end of rgg, are necessary, either by direct involvement in binding or by indirectly affecting secondary structure, for Rgg to increase glucosyltransferase activity. Surprisingly, the presence of the rgg promoter upstream of this 36-bp region significantly increased the effects of plasmid-borne rgg. Implications for glucosyltransferase regulation and applicability to other rgg-like determinants are considered.