Cell-to-cell channels composed of connexin44 and connexin50 were purified from plasma membranes of calf and fetal bovine lenses. The channels were treated with the nonionic detergents octyl-beta-D-glucopyranoside and decyl-beta-D-maltopyranoside, and the channel/detergent complexes purified by ion and gel filtration column chromatography. In negative staining, the channels appeared as annuli 11 +/- 0.6 nm (s.d., n = 105) in diameter and as 16 +/- 0.8 nm (s.d., n = 96) long particles which corresponded to top and side views of 'complete' cell-to-cell channels. The purified cell-to-cell channels were composed principally of a protein, called MP70, that appeared as a diffuse 55-75 kDa band in SDS-PAGE. Dephosphorylation with alkaline phosphatase transformed the diffuse 55-75 kDa band into two distinct bands of almost equal intensity. Immunoblotting showed the bands to be connexin44 and connexin50, respectively. The antibodies also recognized weaker bands composed of the unphosphorylated form of both connexins. The connexins appear to be processed independently 'in vivo'. The unphosphorylated form of connexin50 was present in channels and membranes from fetal, calf and adult bovine lenses, while unphosphorylated connexin44 only in channels purified from fetal lenses. Therefore, lens cell-to-cell channels are composed principally of equal amounts of phosphorylated connexins 44 and 50 that appear to be assembled in the same channel ('hybrid').