We report heritable threefold differences in both larval and pupal esterase 6 activity among 17 isoallelic lines of D. melanogaster extracted from a natural population. The activity differences in the two stages are only weakly correlated with each other or with previously determined values for esterase 6 activity in adults of these lines. The pre-adult activity variation is also unrelated to polymorphisms among the lines for six esterase 6 allozymes and six restriction sites in a region encompassing the esterase 6 coding DNA and the first kbp of 5' flanking DNA. However, two insertions, of 8.0 and 6.8 kbp, located about 1.4 kbp 5' of the esterase 6 coding region are associated with low activity in larvae and, to a lesser extent, in pupae, albeit not in adults. Restriction mapping reveals similarity between the 8.0 kbp insert and the 7.4 kbp retrotransposon 17.6. The differences in larval activity among lines are positively correlated with fitness as assessed from assays of pre-adult viability and development time but no significant associations between pupal esterase 6 activity and these measures are detected. Some effects of esterase 6 allozyme differences are also found for viability and development time but these effects could be explained by linkage disequilibrium between the 8.0 kbp insert and the EST6-9 allozyme.