BACKGROUND: There are different activating salt solutions for sperm induction of rainbow trout (Oncorhynchus mykiss) under artificial spawning condition, although some uncertainties are associated with the performance. OBJECTIVES: This study was conducted to consider the effects of different concentration of calcium and magnesium ions and change of pH in the Billard salt solution on sperm motility of rainbow trout. METHODS: Four-year old broodstocks (n=10) of rainbow trout were randomly selected and adapted to the same experimental condition for two weeks. Experimental solutions were also prepared in triplicate by separately adding 1, 2, and 3 mmol calcium and magnesium to the Billard solution. Another three solutions were also prepared with pH adjustment from 9.0 in the Billard solution to 8.0, 8.5, and 9.5. The broodstocks were then transported to the hatchery and artificially spawned. The collected sperm from all broodstocks were mixed for eliminating the effect of genetically differences between the fishes. Finally, the rainbow trout sperm mobility in the experimental treatments was determined in triplicate and compared by the Billard solution as the control treatment. RESULTS: Sperm mobility of rainbow trout in all treatments was significantly more than the control (p<0.01). The maximum mobility with 105.0±6.7 s was obtained in the 1 mmol calcium treatment, while the lowest level was found in the Billard treatment (31.33±4.50 s). Between the experimental treatments, calcium had the most effect on the sperm mobility of rainbow trout, although no significant differences was observed on the sperm mobility between 3 mmol calcium and 3 mmol magnesium treatments (p<0.01). A downward trend in sperm mobility was found by increasing the pH of activating solutions with the highest and lowest sperm mobility in the pH of 8 and 9.5 treatments, respectively. CONCLUSIONS: The findings of the present study demonstrated that modification of activating salt solutions using each doubly charged ions of calcium and magnesium and the pH adjustment could improve the reproduction efficiency and hatching rate in the hatcheries of rainbow trout.