Neurotrophin 4/5 (NT4/5) is a member of the neurotrophin family known to exert survival and other effects on a variety of neurons including those within the motor, sensory and central populations. Although mRNA(NT4/5) has been found in various effector tissues of the rat and human, the concentration of NT4/5 protein in tissues has not been reported previously due to lack of suitable methodology. We present here a quantitative two-site enzyme-linked immunosorbent assay for the estimation of NT4/5 in pre- and postnatal rat tissues. The assay was performed using a combination of polyclonal and monoclonal antibodies to recombinant human NT4/5. Tissue samples were extracted at neutral pH. Results show that the assay is highly specific for NT4/5 with a sensitivity of 1 pg/ml, and reproducible with intra- and inter-assay variation coefficients of 3.0 and 6.3%, respectively. NT4/5 was found in most embryonic tissues examined at gestation day 17 and 21, but was rarely detectable in postnatal tissues, with the notable exception of the testis. The availability of an immunoassay for the estimation of NT4/5 protein in rat tissues should contribute to the understanding of the physiology of this little understood neurotrophic factor.