A specific and very sensitive dot-immunobinding assay for the detection and enumeration of the bioleaching microorganism Thiobacillus ferrooxidans was developed. Nitrocellulose spotted with samples was incubated with polyclonal antisera against whole T. ferrooxidans cells and then in I-labeled protein A or I-labeled goat antirabbit immunoglobulin G; incubation was followed by autoradiography. Since a minimum of 10 cells per dot could be detected, the method offers the possibility of simultaneous processing of numerous samples in a short time to monitor the levels of T. ferrooxidans in bioleaching operations.