M. lepraemurium grow well in a Balb/c 3T3 recloned cell line (A31). In monolayer culture, the average generation time of M. lepraemurium in A31 cells was 5.3 to 9.4 days at 37 degrees C. A31 cells are very sensitive to infection with M. lepraemurium. Bacterial increases were readily apparent 30 days after inoculating 2 X 10(5) A31 cells in monolayer culture with only six bacilli. The intracellular bacilli were well transferred without apparent losses by host cell transfer. The growth of intracellular bacilli was inhibited by streptomycin 100 micrograms/ml, clindamycin 25 micrograms/ml, INH 5 micrograms/ml, and rifampin 5 micrograms/ml. When streptomycin or clindamycin was removed from the culture medium after 41 days of treatment and the cultivation continued in drug-free medium, the intracellular bacilli began to multiply once more without a lag period. When the intracellular bacilli were treated with INH for 35 days or rifampin for ten days, growth resumed, but only after lag periods after removal of these drugs. We utilized agar suspension techniques for the cultivation of host cells M. lepraemurium because normal cells or transformed cells ceased undergoing cell division and remained healthy for long periods of time in agar medium. M. lepraemurium grew well in A31, A31 transformed by polyoma virus, nude mouse foot pad, chick embryo, and human neuroblastoma cells, utilizing the agar suspension technique. The agar suspension cell culture method should provide useful clues for the cultivation of M. leprae.