The inheritance of Drosophila melanogaster larval pupation behaviour is investigated in sixteen reciprocal crosses between field collected lines. These lines were made isogenic for the two major autosomes enabling the data to be analyzed using contrast analysis of variance and biometrical genetic analysis. Results of both analyses showed that the trait “pupation distance”, the distance larvae pupate from food, fits a simple additive model of inheritance with no dominance. A chromosomal analysis showed that both the second and third chromosomes act additively on pupation distance and that the third pair of chromosomes had a much larger effect than the second. Significant variability exists in the distance D. melanogaster larvae pupate from fruit in nature. This phenotypic variation results from both heritable variation and variation from environmental sources. When the moisture content of the environment surrounding food is modified, gene by environment interactions also contribute to variation in the phenotype. Selective pressures which may act on larval differences in pupation site choice are discussed.