The complete life cycle of Pneumocystis carinii has not been defined, but accumulating evidence suggests that the mammalian host may acquire this organism early in life. In the present study, the initial time of P. carinii acquisition was determined in rats by amplification of P. carinii DNA in oral swabs from seven sets of pups and dams and from fetal tissue obtained by cesarean section of three gravid female rats. DNA extracted from all samples was amplified by using PCR primers directed to the P. carinii mitochondrial large subunit rRNA. Amplicons were produced from 80% (28 of 35) of pups within 2 h after birth; from 97% (34 of 35) after 24 h, and in all of the serially sampled pups by 48 h. No P. carinii amplicons were produced from 48 fetuses or their placentae taken by cesarean section. Thus, P. carinii is acquired almost immediately after birth, and placental transmission occurs rarely, if ever, in rats.