During the prespawning and spawning season experimental groups of +2 year male and female brown trout, Salmo trutta, were kept under natural photoperiod and at three temperature regimes, a naturally fluctuating one with an average temperature of 7.4 ± 4.6°C as typical for alpine and prealpine river systems (T1), a naturally fluctuating one elevated for circa 5°C to 12.4 ± 5.3°C (T2), and a constant one of 9.6 ± 0.8°C (T3). The effect of the three temperature regimes on the timing of spermiation and ovulation, on the maturation rate of males and females and on physiological and biochemical parameters of spermatozoa and oocytes were investigated. T1 was the optimal one for maturation of males and females. Under these conditions >70% of males produced semen of high quality (defined by a volume >3.5 mL, a motility rate >65%, a swimming velocity >135 µm/sec, and a fertility >65%) for a period of 4 weeks. Females ovulated synchronously and the oocytes were of high quality, too (fertility >80%). In T2 the peak in the percentage of mature males was delayed and shortened, the percentage of spermatozoa with DNA damages increased, and peroxidase and lysozyme activity decreased which are indicative for a decrease in semen quality. In females the time point of ovulation was delayed, the fertility of oocytes was reduced, and their phospholipid and free fatty acids levels were decreased. In T3 maturation of fish was not synchronized. However, no negative effect on gamete quality was observed. J. Exp. Zool. 319A:138–148, 2013. © 2013 Wiley Periodicals, Inc.