The formation and reversion of spheroplasts of the diaminopimelic acid-auxotrophic mutant Escherichia coli K 12, 335, DAP-, R+TEM in a medium lacking diaminopimelic acid have been investigated by microphotography: During their development from rod form cells to spheroplasts cells on slide-surface-agar preparations underwent two successive cell divisions in the course of which the cells retained their rod form. The cells formed by these divisions partitioned into a varying number of spheroplasts of different size. The reversion of spheroplasts to rod form cells, started by the addition of diaminopimelic acid showed two characteristic steps: Each spheroplast partitioned again into several spheroplast-like cell bodies; most of them reverted directly to rod form cells. 2. The release of the R-factor mediated periplasmic TEM-beta-lactamase, E. C. 3.4.2.6., into the growth medium during the development of spheroplasts attained more than 50% of the entire TEM-beta=lactamase activity. The spheroplasts showed a multiple enhancement of TEM-beta-lactamase activity per mg cell protein compared with rod form cells.