Transposon (Tn 916) mutagenesis was employed to identify genes in group A streptococcus (GAS) that are involved in bacterial internalization by epithelial cells. One mutant displayed significantly reduced internalization efficiency and was therefore selected for further characterization. The mutant harbored a single Tn 916 insertion in csr, a genetic locus encoding a two-component regulatory system. Mutations in csr were found to derepress hyaluronic acid (HA) capsule synthesis. Since capsule expression has been previously reported to interfere with internalization of GAS, it was possible that the transposon exerted its inhibitory effect either by derepression of capsule synthesis, or by another mechanism. To study the effect of the csr mutation on bacterial internalization, isogenic mutants deficient in either csrR, hasA or both were generated. The hasA mutant adhered to and internalized into HEp-2 cells significantly better than the parent and the csrR mutant strains. The internalization efficiency of the double mutant (csrR(-)/hasA(-)) was reduced by seven-fold compared to that of the hasA mutant. These findings suggest that csrR affects streptococcal entry by modulating capsule expression as well as by another, yet unknown, mechanism.