Burkholderia cepacia has emerged as an important pulmonary pathogen in immunocompromised patients and in patients with cystic fibrosis (CF). Little is known about the virulence factors and pathogenesis of B. cepacia, although the persistent and sometimes invasive infections caused by B. cepacia suggest that the organism possesses mechanisms for both cellular invasion and evasion of the host immune response. In this study, cultured human cells were used to analyze the invasion and intracellular survival of B. cepacia J2315, a highly transmissible clinical isolate responsible for morbidity and mortality in CF patients. Quantitative invasion and intracellular growth assays demonstrated that B. cepacia J2315 was able to enter, survive, and replicate intracellularly in U937-derived macrophages and A549 pulmonary epithelial cells. Transmission electron microscopy of infected macrophages confirmed the presence of intracellular B. cepacia and showed that intracellular bacteria were contained within membrane-bound vacuoles. An environmental isolate of B. cepacia, strain J2540, was also examined for its ability to invade and survive intracellularly in cultured human cells. J2540 entered cultured macrophages with an invasion frequency similar to that of the clinical strain, but it was less invasive than the clinical strain in epithelial cells. In marked contrast to the clinical strain, the environmental isolate was unable to survive or replicate intracellularly in either cultured macrophages or epithelial cells. Invasion and intracellular survival may play important roles in the ability of virulent strains of B. cepacia to evade the host immune response and cause persistent infections in CF patients.