An enzyme-inked immunosorbent assay (ELISA) using the phenolic glycolipid-1 (PGL-1) antigen of Mycobacterium leprae and cross-reactive antisera specific for human IgM was developed to detect IgM antibodies to M. leprae in the nine-banded armadillo (Dasypus novemcinctus). Statistical definitions for positive and negative interpretations in the ELISA were developed by screening animals recently captured and experimentally inoculated with M. leprae. The ELISA was shown to have high sensitivity and specificity. Modern day armadillos of central Louisiana were observed to have a PGL-1 antibody prevalence rate as high as 20%, and a clinical disease rate as high as 5%. A retrospective serological survey of 182 armadillos taken in the years 1960–1964 and predating the use of armadillos in leprosy research was used to evaluate the 1968 environmental contamination hypothesis for the origin of M. leprae infections in the wild armadillo. Antibodies to the apparently species-specific PGL-1 antigen were detected in 17 of the samples taken in 1960–1964. Absorption with whole M. leprae, M. intracellulare, M. terrae, M. rhodesiae, M. scrofulaceum, M. diernhoferi, M. kansasii, M. phlei, M. avium, BCG, and 2 new armadillo-derived mycobacterial species showed these antibody reactions to be specific for PGL-1. Apparently, M. leprae was enzootic in armadillos as early as 1961, and original infection of these animals could not have occurred in 1968.