Caterpillars of Cactoblastis cactorum secrete onto the surface of host cactuses droplets of an oily fluid that issues from the orifices of their paired mandibular glands. The fluid contains a series of 2-acyl-1,3-cyclohexanediones that, collectively, have been shown to elicit trail-following behavior from the caterpillars. This study reports the results of bioassays to determine the ability of two specific compounds, previously shown to be prominent components of the mandibular glands of pyralid caterpillars, 4-hydroxy-2-oleoyl-1,3-cyclohexanedione and 2-oleoyl-1,3-cyclohexanedione, to elicit trail-following behavior from the larvae of C. cactorum. Additionally bioassayed were structural fragments of these molecules. The relative effectiveness of the chemicals in eliciting trail following, the effect of varying concentration on the trail-following response, the importance of specific functional groups to the trail-following response, and the threshold sensitivity of the caterpillar to the pheromone were determined. The study showed that while all the tested compounds elicited some degree of trail following, they differed significantly in their effectiveness. The most effective of the compounds was 4-hydroxy-2-oleoyl-1,3-cyclohexanedione, which, on a per unit volume basis, was as effective as whole gland extract. Caterpillars secreted large quantities of fluid from the glands, and the threshold response to 4-hydroxy-2-oleoyl-1,3-cyclohexanedione occurred at a relative high application rate compared to trail pheromones of other social caterpillars and eusocial insects. This and the observation that the trail marker is secreted from the mandibular glands suggests that the use of 2-acyl-1,3-cyclohexanediones as trail markers is secondary, and that these compounds function primarily in some other, as yet undetermined, context.