ABSTRACT The oestrogenic effects of endocrine‐disrupting compounds (EDCs) on animals are irreversible and have the potential to reduce populations. Sensitive methods, such as quantitative PCR (Q‐PCR), are used to detect alterations in gene expression and thereby predict the consequences prior to the onset of irreversible changes. The present study uses Q‐PCR to detect the expression of the oestrogenic receptors and vitellogenin genes in the mosquito fish ( Gambusia yucatana ) in response to exposure to an acetaminophen sample. The fish were exposed to acetaminophen at nominal concentrations of 0.95, 9.5 and 95 mg/L; a positive control with four female fish, a negative control with four male fish and four male fish were exposed to 17ß‐estradiol at a nominal concentration of 10,000 ng/L as a reference control. The exposure period was 96 h. Samples were collected at 24‐h intervals for each concentration, and the fish were euthanized. The liver and gonads were then removed and stored at a temperature of −70°C. Total RNA was isolated from the liver and gonads tissue of the fish. The relative quantification of gene expression of vitellogenin I and II (VTG I, VTG II), oestrogen receptor α and ß (Erα, Erß) and pregnane X receptor (PXR) was then determined by Q‐PCR on a StepONE Q‐PCR instrument using Maximum SYBRGreen/ROX qPCR Master Mix. The results indicate that the expression of transcripts of oestrogenic receptors, PXR and vitellogenin indicate that acetaminophen has an endocrine disruption effect in G. yucatana .