Voltage-operated Ca2+ channels are heteromultimeric proteins. Their structural diversity is caused by several genes encoding homologous subunits and by alternative splicing of single transcripts. Isoforms of alpha1 subunits, which contain the ion conducting pore, have been deduced from each of the six cDNA sequences cloned so far from different species. The isoforms predicted for the alpha1E subunit are structurally related to the primary sequence of the amino terminus, the centre of the subunit (II-III loop), and the carboxy terminus. Mouse and human alpha1E transcripts have been analysed by reverse transcription-polymerase chain reaction and by sequencing of amplified fragments. For the II-III loop three different alpha1E cDNA fragments are amplified from mouse and human brain, showing that isoforms originally predicted from sequence alignment of different species are expressed in a single one. Both predicted alpha1E cDNA fragments of the carboxy terminus are identified in vivo. Two different alpha1E constructs, referring to the major structural difference in the carboxy terminus, were stably transfected in HEK293 cells. The biophysical properties of these cells were compared in order to evaluate the importance in vitro of the carboxy terminal insertion found in vivo. The wild-type alpha1E subunit showed properties, typical for a high-voltage activated Ca2+ channel. The deletion of 43 amino acid residues at the carboxy terminus does not cause significant differences in the current density and the basic biophysical properties. However, a functional difference is suggested, as in embryonic stem cells, differentiated in vitro to neuronal cells, the pattern of transcripts indicative for different alpha1E isoforms changes during development. In human cerebellum the longer alpha1E isoform is expressed predominantly. Although, it has not been possible to assign functional differences to the two alpha1E constructs tested in vitro, the expression pattern of the structurally related isoforms may have functional importance in vivo.