The potential health risks associated with exposure to Stachybotrys charta rum are currently the subject of intense research, however species-specific detection of the fungus using immunoassays is not available. The purpose of this study was to use the Halogen Inununoassay (HIA) to characterize the antibody binding sites of a recently developed S. chartarum monoclonal antibody (mAb 9B4), in pre- and post-germinated conidia. Antigen expression was detected using mAb 9B4 in the IDA. Aerosolized conidia and fungal fragments were collected onto protein binding membranes direct from sporulating in vitro culture plates. To germinate conidia, samples were incubated in high humidity at room temperature for 48 hours. Ungerminated and germinated samples were then laminated with an adhesive coverslip and immunostained by the HIA. The samples were examined by confocal and light microscopy and positive (haloed) particles were expressed as percentages of total spores. The localization of specific mAb 9B4 binding sites in all pre- and post- germinated samples was primarily restricted to the conidiogenous phialides and around phialoconidia. Post-germination, the proportion of phialides releasing antigen remained unchanged (95.3 +/- 1.5) from ungerminated treatments (96.9 +/- 1.1; P=0.384); however there was a significant increase (P<0.0001) in the proportion of germinated spores expressing antigen (53.3 +/- 2.9) compared to ungerminated spores (6.6 +/- 1.1). No immunostaining was detected in either hyphal fragments or hyphae from germinated spores in pre- and post-germinated treatments. This study further supports that mAb 9B4 recognizes a sporulation specific antigen localized in the phialides and phialoconidia that can be used for Stachybotrys-specific immunoassays.