Analysis of 12 prehistoric human paleofecal specimens from the Mammoth Cave System, Kentucky has produced the first estimate of biological sex using fecal material from ancient humans. Accelerator mass spectrometry (AMS) radiocarbon dating indicates that the specimens range in age from ca. 2700 B.P. to 2300 B.P. Dietary contents and steroids were extracted and analyzed. Chromatography and radioimmunoassay were used to measure levels of testosterone and estradiol in both modern fecal reference samples and in ancient feces. Results indicate that all 12 paleofeces were probably deposited by males whose diet included a variety of native crops and wild plants. These preliminary analyses have the potential to revolutionize the investigation of gender difference in diet, health, and nutrition.