The nuclear lamina, a structure closely apposed to the inner nuclear membrane, is believed to provide a framework important for nuclear envelope integrity and interphase chromatin organization. So far, in mammalian and avian species three major constituents of the lamina, lamins A, B, and C, have been identified. These proteins migrate to characteristic positions on two-dimensional gels, lamin B being more acidic than lamins A and C. Here, we show that the composition of the nuclear lamina in avian and mammalian cells is more complex than previously assumed. When analyzed on two-dimensional gels, the major 66-kDa chicken "lamin B" protein can readily be identified. However, an additional 68-kDa protein migrates to a similarly acidic position. Based on the following evidence, both proteins can be considered as two distinct members of the lamin protein family. First, peptide mapping experiments and immunological criteria demonstrate that these two proteins are not related to each other or to lamin A via postsynthetic modifications or precursor-product relationships. Second, as determined by immunocytochemical techniques, both proteins are located exclusively at the nuclear periphery. Third, both proteins display the biochemical properties characteristic of lamin proteins, i.e. they are resistant to extraction of nuclei with nonionic detergents, nucleases, and high salt. Fourth, both proteins are immunologically related to previously characterized lamin proteins: the major 66-kDa chicken "lamin B" protein shares at least two epitopes with lamin A. However, contrary to what current nomenclature might suggest, this 66-kDa chicken "lamin B" protein is not related to rat liver lamin B, but to a minor component of rat liver pore-complex lamina preparations that had not previously been recognized as a lamin protein. Conversely, the minor 68-kDa component of chicken lamina preparations that had not previously been considered to be a lamin protein is immunologically related to rat liver lamin B. Thus, in addition to demonstrating the existence of quantitatively minor lamin proteins in higher vertebrates, our results caution against assigning structural homologies between lamin proteins from different species on the basis of gel electrophoresis analyses.