Studies of the association of rat-origin Pneumocystis carinii with culture cells were performed both to learn more about the role of cells in P. carinii culture and to evaluate additional cell lines in an effort to improve culture methods. Proliferation of trophozoites of P. carinii from rat lung in cultures with six lung cell lines was demonstrated by light microscopic evaluations of both Giemsa-stained and immune-specific-stained culture samples. Scanning electron microscopy and transmission electron microscopy were used to study the organism's interaction with culture cells and demonstrated a close association of P. carinii with cells in cell lines that supported growth. Proliferation with the MVILU line was suboptimal and there was less organism interaction with these cells than with other cell lines that allowed proliferation. Two cell lines evaluated, Chinese Hamster ovary CHOKI and CHOLEKI, did not allow proliferation and had no association of P. carinii with cells. Scanning and transmission electron micrographs demonstrated the close association of organisms with rat fetal lung (RFL), human embryonic lung (HEL), human diploid lung (HFL), and feline embryonic lung (AKD) culture cells. It appears that the association of rat-origin P. carinii with cells is essential for parasite proliferation in short-term culture.