BACKGROUND: Prolonged transport or poor accessibility of blood culture equipment during night time may cause delayed entry of blood culture bottles. The effect of prestorage conditions on time to detection (TTD) for the blood culture was evaluated for the important gram-negative lactose nonfermentative bacteria. METHODS: Three different clinical isolates of Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, and Burkholdera cepacia were diluted to 150 CFU/mL and 15 CFU/mL and inoculated into standard aerobic bottles. These were stored at 25℃ and at 37℃ for 0, 6, 12, 18, and 24 h. They were entered to BacT/Alert 3D Systems (bio-Mérieux Inc.) and TTD was monitored for each condition. RESULTS: At the 150 CFU/mL concentration, P. aeruginosa and A. baumannii showed false-negative for the bottles prestored at 37℃ for 18 h and 24 h, respectively. However, there was no false-negative for S. maltophilia or B. cepacia at any prestorage conditions. There was a significant decrease of TTD for all experimental microorganisms except P. aeruginosa prestored for 24 h either at 25℃ or at 37℃ (P< 0.05). CONCLUSION: Delayed entry may cause false-negative, especially for the high level of bacteremia of P. aeruginosa or A. baumannii when the bottles are stored at 37℃ for ≥18 h. TTD could be reduced by prestorage of the bottles at 37℃ until 12 h without false-negative for nonfermentative bacteria.