We undertook a prospective study to evaluate the accuracy of PCR of serum (aimed at the pneumococcal pneumolysin gene) at detecting pneumococcal infections in infants and children. The assay was positive for all blood and cerebrospinal fluid culture-positive samples and for 38 and 44% of patients with lobar pneumonia and acute otitis media, respectively. It was positive for 17% of healthy controls. There was a marked effect of age on the rate of positivity among healthy controls, with the highest rate (33%) being in 2-year-old children, the age group with the highest rate of nasopharyngeal (NP) carriage; the lowest rate was found among infants <2 months of age (13%) and adults ages 18 to 50 years (0%), age groups with the lowest NP pneumococcal carriage rates. Carriers of pneumococci in the nasopharynges had a higher rate of positivity than noncarriers of pneumococci in the nasopharynges for all groups. Our results suggest that although PCR of serum is a sensitive test for the detection of Streptococcus pneumoniae in sterile fluids, its high rate of positivity for healthy controls, related to NP pneumococcal carriage, might exclude it from being useful in detecting deep-seated pneumococcal infections.