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To describe the development, evaluation and applicability of a complete method for the detection of Toxoplasma gondii in water.The method incorporated concentration of water samples by Al2(SO4)3-flocculation, purification by discontinuous sucrose gradients and detection of toxoplasmic DNA by 18S-rRNA nested PCR. Tap water replicates and natural water samples were seeded with defined numbers of Toxoplasma oocysts and processed for evaluation studies. When applied to environmental samples, the method gave highest detection sensitivities of 100 oocysts in river water and 10 oocysts in well- and sea water. The method was finally applied in 60 water samples of different quality and origin collected over a 14-month period. Toxoplasmic DNA was detected in four samples.The method offers an alternative towards improving current methods that can be used for the detection of Toxoplasma oocysts in environmental water samples.The method in its current form will be helpful for assessment of Toxoplasma contamination in water resources, particularly after outbreak events.