Sewage treatment plant (STP) effluents with primarily domestic inputs are strongly suspected to be an important source of natural and synthetic estrogens contaminating the aquatic environment. Even a few ng/L of some of these substances can provoke reproductive disturbances in riverine fish. The main purpose of this investigation has been that of ascertaining whether activated sludge STPs (ASSTPs) are able to produce significant amounts of free estrogens. For this purpose, we have monitored monthly estriol (E3), estradiol (E2), estrone (E1) and ethinylestradiol (EE2) in influents and effluents of six Roman ASSTPs for five months. To do this, we have developed an original analytical method involving analyte extraction with a Carbograph 4 cartridge and LC coupled with negative turbo ion spray tandem mass spectrometry in the selected reaction monitoring mode. Analyte recovery ranged between 86 and 91%, and limits of quantification were below 1 ng/L. Over five months, inlet concentrations of E3, E2, E1 and EE...