A number of organisms were screened for their ability to produce l-proline. Kurthia catenaforma, which we recently isolated, was selected. A serine-requiring mutant, strain 45, produced about 1.5 times the amount of this amino acid that the parent strain did. In investigations of various media, it was found that approximately 30 ml of l-proline per ml was produced in shaken culture at 30 C in a medium containing glucose, urea, corn steep liquor, casein hydrolysate, l-aspartic acid, and inorganic salts. To study the effect of l-aspartic acid on the production of l-proline, various amino or organic acids were substituted for l-aspartic acid, and the changes during fermentation were investigated. l-Aspartic acid was not replaced by the compounds tested, and this acid appeared to increase growth during the later stages of fermentation with a concurrent increase in the production of l-proline.