Wild type rat lens main intrinsic protein (MIP) and MIP mutated (F73I, F75L) to resemble the glycerol facilitator of Escherichia coli in the region of the NPA1 box were used to investigate the topology of MIP in the membrane of Spodoptera frugiperda (Sf21) cells using the baculovirus expression system and expression in mouse erythroid leukaemia cells (MEL C88). Differential fixation for staining was used, with paraformaldehyde for externally exposed antigenic sites, and acetone for both externally and internally exposed protein antigenic sites. Immunofluorescence using antibodies to synthetic MIP peptides showed that wild type MIP had a six transmembrane topography. The N- and C-termini were intracellular in both expression systems, and both NPA boxes were found to be extracellular. These results show that residues around the NPA1 box can influence the folding of the MIP in the membrane, and provide structural evidence for the poor water transport properties of MIP, as the NPA boxes lie outside the plane of the membrane.