IntroductionOver the past two decades, there has been increasingconcern about the possible impacts of exposure to chem-icals in the environment on endocrine and reproductivesystems in humans and wildlife [1]. To address theseconcerns, national and international programs have beeninitiated to develop new guidelines for the screening andtesting of potential endocrine-disrupting chemicals (EDCs)in vertebrates [2, 3]. The focus of these multi-tieredprograms was to develop in vitro and in vivo assays toidentify and classify chemicals relative to their potentialinteraction with endocrine systems (Tier 1), and then todevelop concentration–response relationships in animalmodels (Tier 2) [2]. In this article we will focus on a newin vitro bioassay to test for the potential of chemicals tointerfere with steroid hormone production. This assay iscurrently being developed as part of Tier 1 of the USEPAEndocrine Disruptor Screening Program and an Organiza-tion for Economic Cooperation and Development (OECD)test method validation program [4].Most of the efforts to develop screening assays forendocrine disruptor testing have been focused on thedevelopment and validation of in vitro hormone receptor-binding assays including the estrogen receptor (ER) and theandrogen receptor (AR) [5–7]. However, it should beacknowledged that there are a number of non-receptor-mediated processes that may also alter endocrine function[8]. These include chemical-induced modulation of theenzymes involved in the production, transformation, orelimination of steroid hormones, which can result inalterations of the absolute and relative concentrations ofhormones in blood and tissues [9]. Often, these non-receptor-mediated effects are caused indirectly via alter-ations of common signal-transduction pathways [10], orthrough direct competitive or non-competitive inhibition ofthe enzyme.Steroid sex hormones are key factors involved in theregulation of reproduction in vertebrates and are alsoinvolved in numerous other processes that are related todevelopment and growth [11]. Thus, chemicals that candisrupt the production of sex steroids may be directlylinked to adverse outcomes for these processes. To addressthe potential issues of exposure to chemicals that may resultin the disturbance of sex steroid homeostasis, therefore,several in vivo test systems have been evaluated. Thesetests include the pubertal male and female rat assays, thefish reproductive screen, and the frog thyroid assay.Furthermore, several in vitro screening methods have beendeveloped including the microsomal aromatase assay and arodent minced-testis assay [2]. Some of these assays suchas the rodent minced-testis have shown some promiseregarding their potential to identify effects on androgenproduction. However, a number of questions have beenraised about the utility of rodent–based explant assays inthat they were found to yield high rates of false positive or