Prolactin (PRL) plays an important role in freshwater (FW) osmoregulation by preventing the loss of ions and the uptake of water in fish. Growth hormone (GH) promotes acclimation to seawater (SW) in several teleosts. We acclimated rainbow trout Oncorhynchus mykiss weighting $68.2{pm}16.6$, $138.3{pm}24$, and $287.5{pm}42.1$ g in separate experiments to SW under slow-acclimation (SSW) or acute-acclimation (ASW) conditions, and then examined the PRL and GH mRNA levels using the real-time quantitative polymerase chain reaction. The PRL mRNA levels in all three experimental groups decreased significantly with both the SSW and ASW treatments, as compared to a control group kept in FW for 30 days. The GH mRNA levels increased with ASW in the largest fish, whereas the levels in the other groups did not change significantly. The mortality rate of the largest fish was lower than for the other groups, whereas the growth rate among the three experimental groups did not differ significantly. The growth rate of the ASW group was highest for the smallest fish. These results suggest that SW acclimation is associated with the gene expression levels of PRL and GH in relatively large rainbow trout. In addition, the fish mortality and growth rate on FW-SW transfer seem to be related to body weight, and the SW acclimation method may be applied to the hatcheries industry.