A new cephalosporin with a highly reactive beta-lactam ring was found to give an immediate color change in the presence of beta-lactamases from many bacteria, including staphylococci, Bacillus species, Enterobacteriaceae, and Pseudomonas. The reaction is confined to organisms producing beta-lactamases, but it is sufficiently sensitive to indicate the presence of this enzyme is small amounts in strains previously considered not to produce it. The compound has an unusual ultraviolet spectrum, and the color change can be followed quantitatively by measuring changes in absorption which occur in the 380- to 500-nm region, where cephalosporins normally have no absorption. The development of color is thought to be a consequence of the beta-lactam ring being unusually highly conjugated with the 3-substituent. Although in the bacteria only beta-lactamases produce this color change, it was found that serum and tissues from experimental animals also rapidly produced the colored breakdown product, which was then excreted in the urine. The mechanism of the mammalian breakdown was considered to be different from that found in bacteria.