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A sensitive and selective LC-MS/MS method with negative electrospray ionization was developed to detect six estrogen hormones from Danube River and three major tributaries (flu, Olt and Arges). The method separates the estrogen hormones in 25 min using a C18 end-capped column (150 x 2.1 mm, 3.5 mu m) kept at 20 degrees C and a mobile phase made of aq. 0.01% NH4OH and MeOH in the ratio 47.5/52.5 (v/v). Elution program allowed baseline separation of the hormones and consisted of an isocratic step (10 min) followed by a slow gradient (2.5%/min) applied in 4 min. Collision energy, fragmentor and capillary voltages were optimized to increase MS sensitivity. Optimization of LC-MS parameters generated extremely low instrumental LOQ values between 0.06 and 1.1 mu g/L. MS detector response was linear in the range 1 divided by 200 mu g/L with R-2 > 0.99 for all hormones. SPE using Oasis HLB cartridges was employed to concentrate target analytes from water samples. Intra-day and inter-day precision (RSD%) was situated between 5.7 divided by 8.9% and 8.4 divided by 12.6%, respectively. Hormone recovery after SPE was good due to internal standard correction with values between 78 divided by 110%. Overall method LOQs were situated between 0.2 and 3.3 ng/L. Only Estrone was found in Danube and its tributaries at levels from LOQ to 3.8 ng/L. Higher levels of Estrone were detected in tributaries when compared to Danube. Also locations downstream major cities showed higher levels than upstream ones.