The reverse transcriptase inhibitor efavirenz (EFV) is widely used in human immunodeficiency virus (HIV) therapy. Knowledge of the plasma and intracellular concentrations of the drug is of prime importance to get further insight into EFV action in vivo and would be useful for therapeutic drug monitoring (TDM). The aim of this study was to develop a sensitive and specific competitive enzyme immunoassay (EIA) for EFV in biological fluids. Two haptens that differed by the position of the linker were synthesized using two different ways and coupled to BSA. Anti-EFV polyclonal antibodies (pAb) were raised in rabbits using the corresponding immunogens. By comparing results obtained with EIA study with those observed with high-performance liquid chromatography (HPLC) we have shown that the position of the linker appears to be crucial for the specificity of the pAb. EIA was then developed in microtitration plates using the most specific pAb. The assay was performed on a minimum of 30 microL of plasma. It showed good precision and efficiency as well as good cross-validation with HPLC. The lowest limit of quantification (LLOQ) was 150 pg mL(-1), i.e., a value at least 10 times lower than those currently achieved using previously described techniques. This EIA should be useful in the clinical laboratory for monitoring patients during antiretroviral therapy especially young children as well as for measuring EFV in intracellular studies requiring lower amounts of biological material.