This study reports an enzyme-linked immunosorbent assay for detecting Bothrops jararaca venom in fluids, employing the sandwich method with biotin/avidin amplification. The assay exhibits high accuracy in correlating optical densities with venom concentrations (r = 0.98), high reproducibility, low background and limited cross-reactivity with venom from other snake genera. Nevertheless, it was unable to distinguish among venoms from different bothropic species. Using this method we evaluated the serum kinetics of Bothrops jararaca venom in C57BL/6 mice. High concentrations were found in serum just 15 min after injection (151 +/- 41 ng/ml; mean +/- S.D.), followed by a progressive fall (102 +/- 46, 74 +/- 39 and 50 +/- 22 ng/ml after 1, 3 and 6 hr respectively), being undetectable by 24 hr. Such serum kinetics indicates a pattern of a rapid absorption of venom from the inoculation site, followed by a slow and progressive drop in its serum levels. This ELISA was a reliable tool in the determination of Bothrops jararaca venom levels in mouse serum, and may become useful in other fields of bothropic venom research.