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Prolactin, the main freshwater adaptation hormone in fish, is also known as hypercalcemic hormone. It regulates calcium uptake in main osmoregulatory organs such as gills and kidney in fish. Prolactin functions of euryhaline fish Gasterosteus aculeatus are not studied fully, because in G. aculeatus prolactin may be also associated with fanning and parental behaviour. This research aimed to study effect of freshwater adaptation on relative mRNA levels of prolactin in brain (including pituitary) and mRNA levels of prolactin receptors a and b in gills and kidney of female G. aculeatus. In experiment, sticklebacks from sea were placed into freshwater or seawater (control) for three days which is the time of full freshwater adaptation. FW adaptation was associated with relative PRL mRNA level increase in brain of female sticklebacks (93,16±2,08, n=5, vs 68,6±6,5, n=10, %mRNA levels, p=0,0224). This confirms that in stickleback, as in other fish, PRL contributes to low salinity conditions. After FW adaptation PRLRa gene expression in gills increased (113,7±4,7, n=6, vs 105,7±1,4, n=13, %mRNA levels, p=0,047), however, there was no difference in PRLRb gene expression in gills between experimental and control groups. After FW adaptation PRLRb gene expression in kidney increased (121,6±6,2, n=5, vs 103,8±3,7, n=12, %mRNA levels, p=0,02), however, there was no difference in PRLRa gene expression in gills between experimental and control groups. This implies different regulation mechanisms of prolactin on its receptors in different osmoregulatory tissues. Thus FW adaptation stimulates PRL gene expression in female stickleback brain, which causes PRLRa gene expression increase in gills and PRLRb gene expression increase in kidney. This may be associated with different regulation of calcium uptake in different tissues which is yet to be studied more precisely.