To evaluate putative adaptive changes underpinning the invasion of freshwater by the Brachyura, this investigation examines anisosmotic extra and isosmotic intracellular osmoregulatory capabilities in Dilocarcinus pagei, a neotropical, hololimnetic crab, including its embryonic and juvenile phases. All ontogenetic stages show a remarkable ability to survive a high salinity medium (25 per thousand, 750 mOsm/kg H2O, 350 mm Na+, 400 mM Cl-). Adults hyper-regulate hemolymph osmolality up to isosmoticity at 744 mOsm kg/H2O (24 per thousand), [Na+] and [Cl-] becoming isoionic at 449 (22 per thousand) and 256 mM (16 per thousand), respectively. Hemolymph (420+/-39 mOsm/kg H2O) and urine (384+/-44 mOsm/kg H2O) are isosmotic in adults held in freshwater, and after 5-days exposure to 25 per thousand (787+/-9 mOsm/kg H2O and 777+/-43 mOs/kg H2O, respectively); D. pagei does not produce dilute urine. Total free amino acid (FAA) concentrations in embryos (14.9+/-1.2), juveniles (32.8+/-0.1) and adult muscle (10.9+/-2.1 mmol/kg wet weight) in freshwater are 30-fold less than in brackish/marine Crustacea, suggesting that FAA constitute a useful parameter to evaluate adaptation to freshwater. On acclimation to 25 per thousand, total FAA increase by approximately 100% in embryos and in adult muscle and nerve tissue and hemolymph, owing to large increases in proline, arginine and/or alanine. However, effective FAA contribution to intracellular osmolality increases only in embryos, from 3 to 4.5%. These findings suggest that gill-based, anisosmotic extracellular regulation has supplanted isosmotic intracellular regulatory mechanisms during the conquest of freshwater by the Brachyura, and indicate that D. pagei may be an old, well-adapted inhabitant of this biotope.