Using a novel methodology, natural ejaculate volumes of 14 male rainbow trout Oncorhynchus mykiss were compared when males were housed with one female (absence of a rival male) or with another male and one female (rival male present). Contrary to theoretical predictions, male ejaculate expenditure was not influenced by the presence of a rival male. Male gape duration was positively correlated with the volume of sperm ejaculated. Release of sperm by the male always preceded release of eggs by the female. Analysis of the timing and duration of ejaculation suggests that males may rely on the timing and proximity of gamete release to enhance fertilization success. These results are discussed in the context of sperm competition theory.