Antimicrobial susceptibility patterns of major bloodstream pathogens from Iran provide essential information regarding the selection of antibiotic therapy for patients with bloodstream infections (BSIs) living in Iran. Unfortunately, data regarding the isolation frequency and antimicrobial susceptibility patterns of endemic BSI pathogens are scarce in Iran. To shed some light on the susceptibility patterns of BSI pathogens endemic to Tehran, Iran, we investigated the antimicrobial susceptibility patterns of 2248 bloodstream isolates from patients in Children's Medical Center (CMC) Hospital in Tehran between January 1996 and December 2000. Microbiology reports of 24600 blood specimens collected from inpatients in CMC Hospital were retrospectively reviewed. Specimen culture, bacterial identification and disk diffusion susceptibility testing were performed according to National Committee for Clinical Laboratory Standards guidelines. Overall, Gram-positive bacteria comprised 72% (1627/2248) of recovered isolates and Gram-negative bacteria comprised 28% (621/2248). Coagulase-negative staphylococci (CoNS) comprised 48.4% of all isolates, followed by Staphylococcus aureus (16.7%) and Klebsiella spp. (8.5%). Among the 621 Gram-negative organisms, Klebsiella spp. (31%) were the most frequently isolated, followed by Escherichia coli (21%) and Pseudomonas aeruginosa (17%). The rates of oxacillin resistance for S. aureus and CoNS isolates were similar (60% versus 61%); however, the rate of S. aureus vancomycin resistance was almost twice that of CoNS resistance (21% versus 11%). Over 55% of S. pneumoniae were resistant to penicillin and co-trimoxazole. Although all isolates of enterococci were susceptible to vancomycin, only 21% were susceptible to gentamicin. Among Gram-negative isolates, amikacin was shown to be very effective, with susceptibility rates of 84%. The susceptibility of Klebsiella spp. to ampicillin and co-trimoxazole was 1% and 39%, respectively. The susceptibility of Klebsiella spp., E. coli and Enterobacter spp. to ceftriaxone was 47%, 86% and 67%, respectively. There were notable differences in the order of the five most common organisms isolated from blood cultures, which can help set priorities for focused control efforts. Our findings highlight the importance of a nationwide surveillance programme to monitor the trends in isolation frequency of bacteria and their antimicrobial resistance patterns throughout Iran.