This study investigated the effects of incubation delay on the rate of recovery of common pediatric pathogens from blood culture bottles. Known concentrations of Streptococcus pneumoniae and Haemophilus influenzae type b (three isolates each) were inoculated into BACTEC NR-6A bottles with 1.0 mL of donor blood. Bottles were subjected to a time delay (zero to six hours) before incubation. The BACTEC NR-660 was used for incubation and measurement of positive conversion. Data were analyzed using chi 2 analysis, Fisher's exact test, logistic regression, and multiple logistic regression, with P < 0.05 considered significant. Immediate incubation yielded positive blood cultures in 88 of 100 and 65 of 70 bottles containing S. pneumoniae and H. influenzae type b, respectively, in the concentration range 1.0 to 9.99 colony-forming units per milliliter (CFU/ml). For each organism, this was the minimal range required to produce a positive culture (P < 0.0001). Bottles inoculated with 1 ml of blood containing organisms in the range of 1.0 to 9.99 CFU/ml were then subjected to incubation delay. The recovery rate of S. pneumoniae significantly (P = 0.0003) decreased from a two-hour delay (57 of 60; 95%) to a three-hour delay (42 of 60; 70%). No significant change in recovery rate was seen in bottles inoculated with H. influenzae type b subjected to similar delays. Delayed incubation (two to six hours) of bottles inoculated with 1.0 ml of blood containing organisms in a concentration range of 1.0 to 9.99 CFU/ml of blood significantly decreases the recovery rate of S. pneumoniae but has no effect on H. influenzae type b.