Cytocidal activity of neocarzinostatin (NCS) was analyzed in vitro by using cultured and ascitic L1210 [mouse leukemia] cells. NCS shows rapid and typical concentration-dependent cytocidal action against L1210 cells. he concentration required for 90% cell-kill (MLD90) and for all 106 cell-kill (MIC/106) was 5 .times. 10-2 and 4 .+-. 10-1 .mu.g/ml, respectively, when L1210/C cells were exposed to NCS at the concentration of 2 .times. 105/ml in RPMI-1640 medium. When L1210/C cells at the concentration of 1.3 .times. 107/ml were exposed to NCS, cytocidal activity of NCS decreased, and MLD90 and MIC/106 increased to 1.75 .times. 10-1 .mu.g/ml (3.4 .times.) and 2.83 .mu.g/ml (6.5 .times.), respectively. When a small fraction of whole BDF1 mouse blood, red blood cells or spleen cells was present in the reaction mixture, cytocidal activity of NCS appeared to decrease. When washed hemorrhagic ascitic L1210 cell suspension was exposed to NCS, 100 times or more concentration of NCS was required for MLD90 or MIC/106. The effect of plasma or serum on cytocidal activity of NCS was minimum. Cytocidal activity of NCS is probably greatly inhibited through contact with tumor cells, blood cells, spleen cells and/or hemorrhagic ascites. This may be a reason why L1210 cells, which show high sensitivity against NCS in vitro, are less sensitive in vivo. To explain these findings, the possibility of inactivation of NCS by the cells or reduction of free active NCS molecules by the binding or adsorption with the cells is discussed. These characteristics in action of NCS should be taken into account in clinical use.