Human urinary bladder carcinoma cells (JTC-32) retain a low alkaline phosphatase activity. Prednisolone or a hypertonic concentration of NaCl caused a moderate increase in the activity (10- to 15-fold of control), but dibutyryl cAMP or butyrate did not. Examination of the combined effect of these four agents revealed that they acted synergistically in any combination. When the cells were incubated with the four agents together, the enzyme activity increased 60- to 250-fold. Serum also contributed to this synergistic increase. These agents slightly inhibited cell growth and protein synthesis. The enzyme induction was completely inhibited by cycloheximide or actinomycin D. The synergistic effect of the four agents on the enzyme activity was also observed in other strains of carcinoma cells, human urinary bladder carcinoma cells (JTC-30) and monkey hepatocarcinoma cells (NCLP-6E). Thus, it is concluded that the coexistence of the four agents provides general and superior conditions for the induction of alkaline phosphatase in cultured carcinoma cells.