Experimental investigations of time-dependent cytotoxicity of preservatives at concentrations commonly used in eyedrops were conducted with Chang's cultured human conjunctival cells exposed to the rest solutions at 4 degrees C, 15 degrees C and 37 degrees C for logarithmic prolonged time in the range of 128 minutes. Cytotoxicity was expressed quantitatively by the exposure time causing 50% cell damage (CDT50) calculated by the Van der Waerden method. Chlorobutanol at 0.2% concentrations showed no cytotoxicity. CDT50 of 0.3% and 0.4% chlorobutanol at 37 degrees C were 30 minutes, 19 seconds and 17 minutes, 47 seconds, respectively, which were more rapid than the estimated CDT50 at 4 degrees C and 15 degrees C. Ethylparaben at 0.05% concentration caused no cell damage. CDT50 of a mixture of 0.2% chlorobutanol and 0.05% ethylparaben was 37 minutes, 38 seconds at 37 degrees C and more rapid than at 4 degrees C and 15 degrees C. CDT50 of 0.007% benzalkonium chloride was 98.9 seconds at 37 degrees C, 94.2 seconds at 15 degrees C and 91 seconds at 4 degrees C.