The cardenolide-based chemical defenses of danaine butterflies vary macrogeographically. This study demonstrates that these defenses also vary both microgeographically and temporally. We sampled 280 queen butterflies (Danaus gilippus) at 11 sites in northern Florida during the summer of 1993 and determined their cardenolide concentrations and thin-layer chromatography profiles. Queens captured in coastal salt marshes and xeric inland sites were low in cardenolide concentration, while those from hydric inland sites had much higher concentrations. Concentrations also varied temporally, especially at the coastal sites. Thin-layer chromatography analyses of wild-captured queens and those reared on local milkweeds indicated that microgeographic and temporal variation in the cardenolide chemistry of the butterflies is mediated by their host plants. The large differences in cardenolide concentrations among queen populations must result in strong differences in palatability spectra to vertebrate predators. This finding has major implications both for interspecific mimicry and for automimicry.