BACKGROUND: Genome size is known to exhibit interspecies differences, but also to vary between populations within a given species and even between individual cells within an organism. Major differences have often been reported and attributed to differences in measurement conditions, in internal controls of genome size, and in the stains used. Flow cytometry using intercalating dyes is the most attractive method for measuring genome size. METHODS: We estimated relative genome size of nuclei from heads of Drosophila melanogaster adult males using a FACScalibur flow cytometer and propidium iodide. RESULTS: We have shown that the genome size estimates depended on the temperature and humidity of the rearing medium and decreased with age in adult flies. There were large differences in genome size estimates between the vials in which the flies were maintained, but only slight variations within the vials, supporting the idea that the size estimate depends on the fly rearing conditions. Changes in the temperature of the solution of head nuclei analyzed by the cytometer also influenced the genome size estimate. CONCLUSIONS: These findings clearly show that the environmental conditions under which the flies were reared influence the genome size estimate, perhaps as a result of a change in the accessibility of the DNA to the fluorochrome. Caution is therefore called for when estimating genome size. Experimental artifact rather than adaptation may account for some of the correlations between genome size and environmental conditions reported in the literature.