A lack of knowledge persists concerning the combination of kinetics on protein and mRNA levels of the most commonly used biomarker for estrogenic influences—vitellogenin (VTG). Consequently, male fathead minnows were exposed to 17α-ethinylestradiol (EE 2) for 35 days, followed by an equally long depuration period in a flow-through system. VTG mRNA levels reached a plateau after 3 days of exposure, which remained stable until 3 days after EE 2 removal. Control levels were re-attained within 7 days of the depuration phase. VTG protein accumulated in the plasma following a two-phased model. The first phase depicting an exponential increase lasted 15 days and was followed by a saturation phase approaching a plateau of approximately 47 mg VTG/ml plasma. Clearance kinetics could be described by a two-compartment open model, with half-lives of 2.17 and 21.32 days for the α- and β-phases, respectively. In addition, a high VTG protein synthesis rate seemed to adversely affect fitness and mortality of the fish.