The purpose of this study was to evaluate the minimum incubation time required to detect positive blood cultures from newborn infants with sepsis. Data were collected retrospectively on seventy-five positive blood cultures from newborn infants in the neonatal intensive care unit of Songklanagarind Hospital. The BacT/Alert Microbial Detection System had been used to culture the samples. Data were obtained retrospectively from the patients' medical records for positive blood cultures. A computer algorithm in the automated blood culture system determined the time to positivity, which was then evaluated for clinically important definite bacterial pathogens, possible bacterial pathogens, fungi and contaminants. Definite bacterial pathogens accounted for 46% (34/74) of the positive blood culture results, possible bacterial pathogens accounted for 39% (29/74), fungi for 7% (5/74) and contaminants for 8% (6/74). The cultures were positive at 24, 36 and 48 hours of incubation in 70.2%, 91.8% and 95.9% respectively. At 36 hours of incubation, the sensitivity, specificity and negative predictive value were 70.3%, 100% and 93.3%, respectively. All cultures growing clinically significant definite bacterial pathogens were positive by 36 hours of incubations, 88% by 24 hours. The cultures had 100% sensitivity, specificity and negative predictive value at 36 hours of incubation. If definite and possible bacterial pathogens were considered, the time to positivity was 71% at 24 hours, 95% at 36 hours and 97% at 48 hours, respectively. The sensitivity, specificity and negative predictive values were 70.3%, 100%, and 93.3%, respectively. Of cultures growing fungi, 80% were positive by 36 hours and all by 48 hours.