To study the topology of Na+,K+-ATPase monoclonal antibodies (MAbs) specific for membrane-bound enzyme were produced. Using immunofluorescence staining of viable cells or smears of a pig kidney embryonic (PKE) cell line, two groups of MAbs were selected, namely those binding to extra- or intracellular portions of the alpha-subunit. The extracellular location of peptide loop 804-841 linking the Vth and VIth intramembrane hydrophobic segments was proved using MAb VG2. Another MAb, IIC9, interacting with PKE cells only after membrane perforation (4% formaldehyde and 0.1% Tween-20), was shown to bind to the hydrophilic loop 868-945. The antigenic determinants recognized by MAb IIC9 and VG2 are located in peptides 887-904 and 810-825, respectively. The C-terminus of the alpha-subunit molecule was positioned on the outer side of the cytoplasmic membrane utilizing affinity-purified antibodies to the synthetic peptide corresponding to fragment 999-1008.