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The asymmetry of the DNA duplex due to polynucleotide strand complementarity could be the molecular basis of cell polarity in spore-forming bacteria. To test this possibility, the relationship of DNA strand segregation to the spore location pattern in chains of sporangia was investigated in Bacillus megaterium. Spores containing one chromosome labelled in one of the complementary strands were formed from cells that had been allowed to segregate pulse-labelled chromosomes in minimal medium at 30°C. A second crop of spores was then formed from cells originating from the labelled spore population. The second generation spores inherited labelled strands from the first spore population by random segregation. In contrast, the patterns of spore positions in sporangial chains were non-random. Furthermore, the non-randomness of patterning was stable and was unaffected by growth temperature (15 to 37 °C) or by enrichment of the minimal medium used in the segregation experiments. Since the pattern of DNA strand segregation is random and the spore location pattern in chains of sporangia is non-random, the asymmetry of the DNA duplex cannot be the determinant of cell polarity.