Arbuscular mycorrhizal fungi (AMF) produce the putative protein glomalin, which is quantified from soil as the operationally defined GRSP (glomalin-related soil protein). GRSP is generally well correlated with soil aggregate water stability, and may hence be a useful integrative indicator of soil “health”. Given the problems with tracing the origin of various fractions of GRSP derived from soils to AMF, we have conducted several studies under conditions of sterile in vitro culture, as opposed to earlier, phenomenological studies describing pool changes under field conditions. Using this approach we showed that glomalin is contained in the mycelium (hyphal wall), rather than being primarily secreted. Other than field phenomenological data, we have very little information about what factors control the production flux of glomalin (in the absence of decomposition). To fill that gap, we here present the development of an in vitro experimental design in the absence of the host (i.e., germinating spores) to screen a variety of environmental factors that potentially control fungal production (as assessed with the monoclonal antibody, MAb32B11) of this protein. Additionally, we report on ongoing biochemical/ molecular characterization of glomalin, which will further inform potential functions of this protein in the life history of AMF.