To elucidate the osmoregulatory mechanisms underpinning the invasion of fresh water by the palaemonid Crustacea, we investigate the contribution of free amino acids (FAA) to intracellular isosmotic regulation in selected ontogenetic stages of two diadromous, neotropical shrimps, Macrobrachium amazonicum and M. olfersi, exposed to fresh water or to saline media. We also evaluate anisosmotic/ionic extracellular regulatory capability in adult M. amazonicum alone; all data for adult M. olfersi are from McNamara et al. (2004). Adult shrimps show similar osmotic and ionic regulatory capabilities, including elevated hemolymph osmolality in fresh water, moderate isosmotic points, hyper-regulatory capability up to 20‰, and good tolerance of saline media. However, the two species rely on brackish water to different degrees to complete their life cycles: while M. olfersi zoeae 1 and 2 survive well in fresh water, those of M. amazonicum die within two hours. Total FAA titers increase significantly over the ontogenetic sequence in both species, independently of salinity exposure, concentrations increasing sharply in M. amazonicum zoeae 1 alone, but steadily from embryos to adult M. olfersi. While total FAA titers increase significantly on transfer of zoeae 1 (+ 43%) and adult (muscle + 72%, gill + 62%) M. amazonicum to elevated salinity (25‰), their effective contribution to hemolymph and intracellular osmolality is unaltered (≈16% in zoea 1, 6-8% in zoea 2 and adult tissues). Total FAA titers in M. olfersi increase in embryos (+ 95%), zoeae 1 (+ 23%) and post larvae (+ 28%), and in adult tissues (muscle + 69%, gill + 110%, nerve + 187%) after salinity exposure. However, effective contribution to intracellular osmolality increases only in embryos (5 to 6%) and adult nervous tissue (6 to 13%). In both species, total FAA increase is due to the most abundant non-essential FAA, glycine, alanine and proline, and arginine. Our analysis shows that diadromous species like M. amazonicum and M. olfersi exhibit lower total FAA titers compared to marine species. Such findings allow a better understanding of the physiological mechanisms underlying the invasion of fresh water by these recent colonizers.