1) The author observed the influences of osmotic tension upon the clinical tissue culture of mouce's spleen. In the hypotonic medium, although the rate of relative growth was higher than in the isotonic medium, the cell density was lower and in the wandering velocity of lymphocytes no significant difference was shown. In the hypertonic medium the rate of relative gromth, the index of cell density and the wandering velocity of lymphocytes were lower than in the normal medium. 2) The author observed the influences of temperature upon the clinical tissue culture of mouce's spleen. The most suitable temperature to culture was from 35℃ to 37℃, and the range of temperature in which culture was possible was from 25℃ to 39℃. At 33℃ and 35℃ the progress of degenerative process was delayed. 3) In order to observe the influences of hydrogen ion concentration of media upon the clinical tissue culture of mouce's spleen, the author cultured by adding the various concentration of hydrochloric acid or sodium hydroxide in the medium. In acid medium the growth was possible even by adding 1/100 N hydrochloric acid, and in alkaline medium the culture was possible even by adding less than 1/500 N sodium hydroxide. All the rate of relative growth, the index of cell density and the wandering velocity of lymphocytes, except the rate of relative growth in the medium adding 1/1000 N hydrochloric acid, were lower especially in high concentration of acid or alkali than in the control medium adding physiological salt solution. 4) According to these results in the clinical tissue culture of spleen, except relatively lower temperature, it was shown that unphysiological physicochemical conditions suppressed or stimulated the growth of tissue culture and were harmful to culture.