A sensitive quantitative bioassay was developed for the measurement of Bacillus thuringiensis Berliner -endotoxin, Cry1Ac, in Bollgard cotton plant tissue. Cotton tissue samples were evaluated in a sensitive Heliothis virescens (F.) growth inhibition bioassay, with Cry1Ac levels being quantified by comparison with concurrently run concentration-response curves using purified Cry1Ac protein. The assay was amenable to large numbers of samples, used small amounts of plant tissue, and avoided some of the concerns associated with immunobased quantitative assays. Field studies were conducted during the 1995 growing season in which Cry1Ac levels were quantified in the Bollgard cotton line 531 (in Coker 312 background). Analyses were conducted to test for the effects of environment (field site differences), plant age, and tissue type on Cry1Ac concentrations. The small foliage of main terminals and the primary, or proximal, fruiting structure from node 9 (9th main stem branch from the bottom of the plant) were sampled at various intervals throughout the growing season at 6 field sites. Mean Cry1Ac levels in terminals and fruiting tissue differed significantly, with terminal foliage levels higher than node 9 fruit. Within each tissue type, significant differences among sites were also seen, suggesting environmental influence on either Cry1Ac production or stability. Mean Cry1Ac values in fruiting structures declined significantly from 57.1 g/g dry weight at 53 d after planting to 6.7 g/g at 116 d after planting. Mean terminal Cry1Ac levels also declined significantly from 163.4 g/g to 34.5 g/g at 53 d after planting and 116 d after planting, respectively. In general, Cry1Ac levels in the fruit and terminals of Bollgard cotton declined steadily as the growing season progressed, with greater activity always occurring in the terminal foliage.