Ankyrins are a family of large membrane associated proteins that link the spectrin-based cytoskeleton to integral membrane proteins, via a domain consisting of twenty four 33-residue repeats. We have cloned and characterized a novel ankyrin isoform which lacks several of these ankyrin repeats and fails to assemble with the peripheral membrane cytoskeleton. Using a PCR product targeted to conserved regions of previously described ankyrins, we isolated clones encoding for a truncated ankyrin isoform from a human kidney cDNA library. Nucleotide sequencing predicts a 116 KDa protein, AnkK, which is expressed as a short 6 Kb message only in kidney, placenta, and muscle. An antibody raised to AnkK recognized a 116 KDa peptide in kidney tissue and MDCK cell lysates, in the Triton-extractable pools (in contrast to the Triton-stable distribution of conventional ankyrins). Indirect immunoflourescence on MDCK cells revealed a surprising punctate staining clustered near and around the nucleus, which closely resembled the Golgi apparatus. In double staining studies, the AnkK immunoreactivity was found to co-localize with β-COP (an established Golgi marker) and withβ-spectrin (which has recently been shown to associate with Golgi membranes in kidney cells). Furthermore, in vitro solution binding assays showed that AnkK directly and specifically interacts withβ-spectrin, with an affinity in the nanomolar range. AnkK thus represents a novel truncated renal and muscle ankyrin isoform that localizes to the Golgi complex and avidly binds β-spectrin in kidney cells. We speculate that it may play a crucial role in organizing and stabilizing the Golgi membranes(much like the larger ankyrins do at the plasma membrane), and in chaperoning nascent or displaced ligands that are destined for the peripheral membrane-cytoskeletal complex.