The aim of the present study was to assess the effects of ammonia and water hardness on endocrine, osmoregulatory and metabolic parameters in silver catfish (Rhamdia quelen). The specimens (60–120 g) were subjected to six treatments in triplicate, combining three levels of un-ionized ammonia (NH3) (0.020 ± 0.008 mg/L [1.17 ± 0.47 μM], 0.180 ± 0.020 mg/L [10.57 ± 1.17 μM] and 0.500 ± 0.007 mg/L [29.36 ± 0.41 μM]) and two levels of water hardness (normal: 25 mg CaCO3/L and high: 120 mg CaCO3/L), and sampled after two exposure times (1 and 5 days post-transfer). Plasma cortisol, metabolites, osmolality and ionic values were determined concomitantly with the mRNA expression levels of different adenohypophyseal hormones (growth hormone, GH; prolactin, PRL; and somatolactin, SL). Previously, full-length PRL and SL as well as β-actin cDNAs from R. quelen were cloned. Exposure to high NH3 levels enhanced plasma cortisol levels in fish held under normal water hardness conditions but not in those kept at the high hardness value. The increase in water hardness did not alter plasma metabolites, whereas it modulated the osmolality and ion changes induced by high NH3 levels. However, this hardness increase did not lead to the decreased GH expression that was observed 5 days after exposure to 0.18 mg/L NH3 in fish held at the normal water hardness level, whereas PRL expression was enhanced after one day of exposure under the increased hardness conditions. Additionally, SL expression decreased in specimens exposed for 5 days to 0.18 mg/L NH3 and maintained at the high water hardness level. The results showed that increasing water hardness attenuated the hormonal parameters evaluated in R. quelen specimens exposed to high NH3 levels, although plasma metabolism do not appear to suffer major changes.