Abstract:A preliminary study of antibiotic production and antibiotic resistance was conducted in Great Onyx Cave in Mammoth Cave National Park, KY, to determine if gypsum (CaSO 4 ·2H 2 O) affects these bacterial activities. The cave crosses through the width of Flint Ridge, and passages under the sandstone caprock are dry with different amounts of gypsum. The Great Kentucky Desert hypothesis posits that gypsum limits the distribution of invertebrates in the central areas of Great Onyx Cave. Twenty-four bacterial isolates were cultivated from swabs and soils. Using three methods (soil crumb, soil crumb with indicator bacteria, and the cross-streak method using isolated bacteria) we did not detect any production of antibiotics. Antibiotic resistance was widespread, with all 24 isolates resistant to a minimum of two antibiotics of seven tested, with three isolates resistant to all. Antibiotic resistance was high and not correlated with depth into the cave or the amount of gypsum. The Great Kentucky Desert hypothesis of the negative effects of gypsum seems to have no impact on bacterial activity.