Owing to the discovery of a less soluble crystalline form (form 2) of cyclosporine (CsA), risks in solubility and physical stability of these formulations need to be revisited. This work focused on understanding the solubility behavior of various CsA forms in different media, including water, castor oil, and selected cosolvent micellar systems. In water, form 2 was approximately 8-9 times less soluble than form 1 (aka. tetragonal dihydrate). In neat nonaqueous solvent, for example, castor oil, form 3 (aka. orthorhombic hydrate) was found to have the lowest solubility and therefore the most stable form. In addition, the solubility-temperature relationship of CsA is complex and solvent-dependent. In aqueous vehicles, retrograde temperature dependence of solubility was observed in aqueous vehicles, that is, the solubility of CsA decreased with temperature, which was attributed to the effect of temperature on the strength of hydrogen bonding interactions; conversely, the solubility of CsA increased with temperature in nonaqueous solvents. In addition, the solubility of these CsA forms was very sensitive to temperature. Temperature-dependent form transformation was also observed in the media studied, with faster form conversion occurring at elevated temperatures. These studies provided key information to support the risk assessment for topical ophthalmic formulation development of CsA.