A highly sensitive and robust method was developed for routine analysis of two progestin metabolites, 17 alpha-hydroxypregnanolone (17OH-Delta 5P) and pregnanediol (PD), and 31 other natural and synthetic steroids and related metabolites (estrogens, androgens, corticosteroids, progestins) in river water, as well as influents and effluents of municipal wastewater treatment plants (WWTP) using HPLC-MS/MS combined with solid-phase extraction. For the various matrixes considered, the optimized method showed satisfactory performance with recoveries of 70-120% for most of target steroids. The method detection limits (MDLs) ranged from 0.01 to 3 ng/L for river water, 0.02 to 10 ng/L for WWTP effluents, and 0.1 to 40 ng/L for influents with good linearity and reproducibility. The developed method was successfully applied for the analysis of steroids in rivers and WWTP influent and effluents. WWTP influents concentrations of 17OH-Delta 5P and PD were 51-256ng/L and upto 400ng/L, respectively, along with androstenedione (concentration range: 38-220 ng/L), testosterone (11-26 ng/L), estrone (2.3-37 ng/L), 17 beta-estradiol (N.D.-8.7 ng/L), 17 alpha-hydroxyprogesterone (N.D.-66 ng/L), medroxyprogesterone acetate (N.D.-5.3 ng/L), and progesterone (2.0-22 ng/L), while only androstenedione (ADD), estrone (E1), and estriol (E3) were detected in effluent with concentrations ranging up to 1.7 ng/L, 0.90 ng/L and 0.8 ng/L, respectively. In river water samples, only ADD and E1 were detected with concentrations up to 1.0 ng/L and 0.91 ng/L. Our procedure represents the first method for analyzing 17OH-Delta 5P and PD in environmental samples along with a large series of steroids. (C) 2017 Elsevier B.V. All rights reserved.