We investigated growth conditions that regulate the ability of group B Streptococcus (GBS) to attach to, invade, and translocate through polarized human respiratory epithelial cells (RECs). GBS grown in a chemostat at a fast cell mass doubling time (t(d) = 1.8 h), invaded RECs from both the apical and basolateral surfaces in higher numbers compared with those held at a t(d) = 11 h. With the exception of adherence from the basolateral surface, GBS reached peak adherence to, invasion of, and translocation through RECs when held at the fast t(d) with 15% dissolved oxygen compared with 0% dissolved oxygen. Growth rate and oxygen level strongly influence the interaction of GBS with polarized RECs and likely GBS pathogenicity.