The neuronal microtubule-associated protein, tau, is expressed as a set of isoforms containing either three or four tandemly repeated 31-amino-acid motifs in the C-terminal half of the molecule that can bind to microtubules. Three-repeat forms are the only ones expressed early in development. A single three-repeat isoform of tau has been stably expressed in non-neuronal cells which do not express endogenous tau. Chinese hamster ovary (CHO) cells were transfected with a full-length cDNA coding for the foetal form of human tau cloned downstream of the simian virus 40 (SV40) promoter, and a cell line constitutively expressing tau, CHO[pSVtau3], was isolated. Double-label immunofluorescence microscopy reveals that tau co-localizes with the microtubular network of normal or taxol-treated CHO[pSVtau3] cells, without inducing any dramatic change in cell morphology. Tau is expressed in CHO[pSVtau3] cells as three bands in SDS/PAGE recognized by antibodies to tau, the slow-migrating tau species being the most abundant. Tau also appears as three bands in a heat-stable fraction from CHO[pSVtau3] cells, but a single band of enhanced immunoreactivity is detected following treatment of this fraction with alkaline phosphatase. This single band co-migrates with the fast-migrating band of untreated fractions or whole-cell extracts. In conclusion, a three-repeat isoform of tau is capable of binding to microtubules in transfected non-neuronal cells; furthermore, in this system, the protein is phosphorylated in at least two different states inducing a reduced electrophoretic mobility.