Measurement of steroids that are released into the water via the gills has previously been shown to be an effective way of studying the reproductive endocrinology of the male three-spined stickleback Gasterosteus aculeatus without having to kill the fish. In the present paper, a previous observation on the existence of a compound other than 11-ketotestosterone (11-KT) in water, which cross-reacted in the 11-KT radioimmunoassay was repeated. The amounts of this compound, however, were not sufficient to warrant a separation step prior to carrying out assay. The lack of association between androstenedione levels in water and those in plasma was also confirmed. For the first time, the amounts of testosterone released into the water were shown to be positively correlated with the amounts in plasma, the sampling procedure (placing the fish for 30 min in 50 ml water) had no effect on the rate of release of cortisol but caused a rapid drop in the rate of release of 11-KT (which means that the fish should not be sampled twice in short succession), physical interaction between two nesting males (which was accompanied by aggression) significantly increased the rate of release of 11-KT, androstenedione and testosterone (but not of cortisol) and the rate of release of 11-KT was at its maximum between 2 and 4 h after exposure.