Sarotherodon melanotheron is one of the most euryhaline teleosts able to withstand variations in environmental salinity ranging from freshwater (FW) to 130‰ hyper-saline waters (HSW). Although significant progress has been made in exploring the cellular and molecular changes that accompany salinity adaptation in teleosts, little is known about the effects of long-term acclimation to HSW. We sought to identify in this tilapia species the genes whose transcription is induced by long-term acclimation either to HSW or FW. Two subtractive cDNA libraries were made from gills of fish acclimated for 45 days to either condition, with 320 partial cDNA sequences encoding proteins potentially involved in the response to the two salinity extremes. The ESTs comparisons with genomic databases allowed putative functions to be attributed to 197 of these genes. The suppression subtractive hybridisation (SSH) results were validated by Real-time PCR for 13 candidate genes having presumably a role in osmoregulation, supplemented by Na(+), K(+)-ATPase α-subunit and carbonic anhydrase, two genes known to be implicated in this function. In fish acclimated to both salinity extremes, the functional category of cellular process was the predominant one, which may indicate high cellular turnover rates in FW and HSW-adapted fish. The acclimation to FW and HSW also appeared to trigger the expression of genes involved in transport activity, biological regulation and metabolic processes, at a higher level in fish acclimated to HSW, suggesting higher metabolic activity in this situation. These results are a first step towards the identification of key molecular processes involved in the fish acclimation to extreme salinities.