Non-classical MHC class I HLA-E, -F, and -G molecules differ from classical class I histocompatibility antigens by specific patterns of transcription, protein expression, and immunological functions. Restriction of the expression pattern of these non-classical antigens may play a key role in modulation of immune responses during pregnancy and diseases but remains to be additionally defined. A specific component of the second International Conference on HLA-G and the 13th HLA-G Histocompatibility Workshop will be dedicated to the analysis of transcription and expression of non-classical class I genes in normal and pathological tissues. In a first step, referred to as the preworkshop, we here report the analysis and conclusions of a working group which was constituted to gather and validate optimal reagents and protocols allowing RT-PCR analysis of HLA-E, -F, -G transcript levels and flow cytometry and immunochemistry analysis of HLA-G expression in cells and tissues. As a result of this work, use of specific primers and probes detecting alternative transcripts of HLA-E, -F, and G have been validated in transfected cells expressing differential pattern of HLA class I antigens. Analysis of the specificity and affinity of collected antibodies has allowed definition of reagents to be proposed for immunochemistry and flow cytometry analysis of HLA-G expression in normal and pathological tissues during the workshop. This work has allowed constitution of an extended workshop group which is now initiating analysis of non-classical class I transcription and expression in various cells and tissues, a collective contribution that will additionally refine our view of the expression of these antigens in normal and pathological situations.