Although Streptococcus pneumoniae is the leading cause of community-acquired pneumonia in humans, the mechanism whereby the organism penetrates lung tissue is poorly understood. In the present study we have examined the capacity of pneumococci to penetrate A549 cells, a human lung alveolar carcinoma (type II pneumocyte) cell line. Not all clinical S. pneumoniae isolates initially tested were capable of penetration of the cells, as judged by resistance to extracellular antibiotics. The presence of a polysaccharide capsule also significantly reduced the capacity to both adhere to and penetrate A549 cells. Electron micrographs showed the presence of pneumococci enclosed within vacuoles of intact A549 cells, but bacteria were also seen free in the cytoplasm of damaged cells. Ongoing bacterial DNA, RNA, or protein synthesis was not essential for uptake of pneumococci by A549 cells, and uptake was not diminished by pretreatment of the pneumococci with trypsin. However, inhibition of A549 microfilament assembly with cytochalasin D abolished the phenomenon.