Four strains of euryhaline bacteria belonging to the genus Halomonas were tested for their response to a range of temperatures (2, 13, and 30°C), hydrostatic pressures (0.1, 7.5, 15, 25, 35, 45, and 55 MPa), and salinities (4, 11, and 17% total salts). The isolates were psychrotolerant, halophilic to moderately halophilic, and piezotolerant, growing fastest at 30°C, 0.1 MPa, and 4% total salts. Little or no growth occurred at the highest hydrostatic pressures tested, an effect that was more pronounced with decreasing temperatures. Growth curves suggested that the Halomonas strains tested would grow well in cool to warm hydrothermal-vent and associated subseafloor habitats, but poorly or not at all under cold deep-sea conditions. The intermediate salinity tested enhanced growth under certain high-hydrostatic-pressure and low-temperature conditions, highlighting a synergistic effect on growth for these combined stresses. Phospholipid profiles obtained at 30°C indicated that hydrostatic pressure exerted the dominant control on the degree of lipid saturation, although elevated salinity slightly mitigated the increased degree of lipid unsaturation caused by increased hydrostatic pressure. Profiles of cytosolic and membrane proteins of Halomonas axialensis and H. hydrothermalis performed at 30°C under various salinities and hydrostatic pressure conditions indicated several hydrostatic pressure and salinity effects, including proteins whose expression was induced by either an elevated salinity or hydrostatic pressure, but not by a combination of the two. The interplay between salinity and hydrostatic pressure on microbial growth and physiology suggests that adaptations to hydrostatic pressure and possibly other stresses may partially explain the euryhaline phenotype of members of the genus Halomonas living in deep-sea environments.Euryhaline bacteria, which can grow over an extremely wide salt range, are nearly ubiquitous in marine environments and are often cultured from deep-sea sediments and hydrothermal vents (32,52,70,76). These microorganisms are also abundant; bacteria capable of growth on media with 17% total salts, including primarily members of the genera Halomonas and Marinobacter, were found to comprise a remarkably high percentage (up to Ͼ10%) of the total microbial community in hydrothermal-vent habitats and the overlying water column in the North and South Pacific ocean (32). Several recently characterized Halomonas strains isolated from low-temperature hydrothermal fluids, hydrothermal plumes, and sulfide rock, including those from 1:50 to 1:500 fluid dilution enrichments, were found to have a minimum growth temperature of Ϫ1 to 2°C at 0.1 MPa and 4% total salts (33), similar to Antarctic Halomonas isolates (38,58,76). This low minimum temperature for growth closely matches the temperature of the deep sea below 1,500 m (and shallower towards the poles) and leaves open the question of whether Halomonas strains collected from the deep sea, despite their numerical significance, are able to grow in situ in ...