The effect of chromosome age on segregation during sporulation was investigated. Vegetative cells of Bacillus megaterium were labeled with [Me-3H]thymine and then were grown at 30 degrees C in nonradioactive medium for various times before being allowed to sporulate. The ratio of the amount of label in sporal DNA to that in sporangial DNA, obtained after minor correction for the sporulation frequency, remained essentially constant as the postlabeling growth period was increased from one to seven generations. The spores were preferentially located at the older poles of sporangia, i.e. the poles formed by divisions occurring prior to those forming the sporangia. Therefore, it seems that old (labeled) chromosomes segregate randomly with respect to both the morphological and genealogical polarities of sporangia. Examination of total cell lysates by dye-buoyant density gradient centrifugation revealed the presence of covalently closed circular DNA from cells grown at 37 degrees C, but none was obtained from cells grown at 30 degrees C. Thus, possible interference by large amounts of extrachromosomal DNA in the determination of the chromosomal segregation pattern is unlikely.