To determine the effect of different nitrogen sources and osmotic regulators on minimal growth of Habanero pepper (Capsicum chinense Jacq.) germplasm for in vitro conservation, different concentrations of nitrate, sucrose, mannitol, and sorbitol were evaluated. The micropropagation system based on Santana-Buzzy et al. (2006) culture medium was modified in its nitrate concentrations: reduced to 50% and increased to 150%, and osmoregulators were added to the basal culture media: sucrose (6% and 8%), mannitol (2%, 4%, and 8%), or sorbitol (2%, 4%, and 8%). The apical meristems of germinated plants were cultivated in the different treatments for 35 weeks without subculture. Results have demonstrated that mannitol at 2% had the better effect on minimal growth of the plantlets and did not affect the plant physiology and quality. The plantlets remained small in size, turgent, with green leaves and stems and looked like normal plants until to the end of the evaluation period. Changes in nitrogen media concentration did not prove to be adequate for conserving because they affected the plantlet quality (they became chlorotic). The presence of sorbitol and high osmolite concentrations induced minimal growth but reduced the plant quality. Sucrose at mid or low concentrations did not induce minimal growth.