The nucleoside analogue, 1-(2-deoxy-2-fluoro-1-D-arabinofuranosyl)-5-iodouracil (FIAU) is a substrate for thymidine kinase (TK), which is commonly expressed in bacteria. It is currently being investigated in clinical studies as an in vivo bacterial infection detection agent. In developing countries where imaging facilities are not readily available, deploying such technology can be a big hurdle. However, a portable ex vivo system might provide a good alternative. In an in vitro system, [(125)I]-FIAU incubated with bacteria is phosphorylated by TK, and is trapped within the bacteria, which can be detected by radioscintography. The suitability of this agent to be utilized as part of an ex vivo bacterial detection system was evaluated. In the first part of this report, the optimization of the incubation and detection condition using E. coli as a test case is described. Samples were incubated in a growth promoting medium containing the label, then after filtering and washing, the amount of radioactivity trapped on the filter was quantitated by a scintillation counter. As a proof of concept demonstration, blinded urine samples from urinary tract infection (UTI) patients and normal donors were tested in the FIAU system. Of the 13 UTI positive and 15 normal urine samples tested, there were 2 false negatives and 1 false positive, respectively. Potential explanations for the false positive and negatives as well as the commercialization possibility of this system will be discussed.