Forty-five isolates of Streptococcus pneumoniae were inoculated on Dorset egg and supplemented Columbia agar base media, incubated overnight at 37 degrees C, and then kept at room temperature (RT; 21 degrees C) or 4 degrees C. Long-term viability was best at RT for both media, with all isolates remaining viable on Dorset egg medium for 44 days; viability was 90 and 57% on Columbia agar base medium after 7 and 30 days. We recommend the use of Dorset egg medium for the maintenance of pneumococci at RT.