BACKGROUND It is important to identify all circulating metabolites, including free fluoride, for accurate pharmacokinetic modeling of [(18)F]-labeled radiotracers. We sought to determine the most efficient method to detect and quantify low levels of free [(18)F]fluoride in biological samples. METHODS Low levels of [(18)F]fluoride were analyzed using two methods: (A) an ion-exchange cartridge and gamma counting, and (B) radio-HPLC, to compare the detection limits of these two analytical methods. Twenty microliters of [(18)F]fluoride solution was loaded onto an ion-exchange cartridge, then eluted with 20% MeCN/water (5 ml) and radioactivity trapped in the cartridge counted on a gamma counter. [(18)F]Fluoride was also determined in plasma and urine from mice injected with [(18)F]-labeled thymidine analogues using Method A. RESULTS The detection sensitivity of Method A was 9.4-fold higher than that of Method B (0.075±0.004 vs. 0.71±0.02 nCi). With Method A, [(18)F]fluoride was determined in plasma for [(18)F]FLT, [(18)F]FMAU, [(18)F]FEAU and N(3)-[(18)F]FPrT as 1.4±0.31% (n=4), 0.17±0.49% (n=3), 4.88±1.62% (n=3) and 12.94±0.48% (n=4), respectively. The amount of [(18)F]fluoride determined in the urine was 11.49±1.60% (n=4) from [(18)F]FLT, 5.36±2.34% (n=3) from [(18)F]FMAU, 13.57±1.96% (n=3) from [(18)F]FEAU and 11.19±1.98% (n=4) from N(3)-[(18)F]FPrT. CONCLUSIONS Low levels of [(18)F]fluoride in biological samples can be detected and quantified using an ion-exchange cartridge and gamma counting. This methodology is simple, accurate and superior to the standard use of radio-HPLC on a C(18) column for metabolite analysis, and it should be useful in pharmacokinetic modeling for animal imaging studies using an [(18)F]-labeled radiotracer and PET.