Induction to cell wall-deficient bacteria has been suggested to be a cause of intractable and opportunistic infection after chemotherapy. Spheroplast formation by beta-lactam antibiotic in not so high osmotic pressure was investigated in six species of gram-negative bacteria. Some species of gram-negative bacteria were induced to form spheroplast at a high rate by 1:10 concentration of ceftizoxime in the presence of Ca2+ or Mg2+. Especially in 38% of Proteus mirabilis and P. vulgaris, more than 40% of the original cells were induced to form spheroplast by ceftizoxime in a medium supplemented with 40 mM Ca2+. The same formation rate was also found in 22% of Serratia marcescens. Formation rates in the drug sensitive strains of S. marcescens were enhanced as the drug concentration increased. Ca2+ was more effective in spheroplast formation than Mg2+.