To further characterize the interaction of cytoplasmic intermediate filament (cIF) proteins with supercoiled (sc)DNA, and to support their potential function as complementary nuclear matrix proteins, the type III IF proteins vimentin, glial fibrillary acidic protein, and desmin were analyzed for their capacities to interact with supercoiled plasmids containing a bent mouse gamma-satellite insert or inserts capable of non-B-DNA transitions into triplex, Z, and cruciform DNA, that is, DNA conformations typically bound by nuclear matrices. While agarose gel electrophoresis revealed a rough correlation between the superhelical density of the plasmids and their affinity for cIF proteins as well as cIF protein-mediated protection of the plasmid inserts from S1 nucleolytic cleavage, electron microscopy disclosed binding of the cIF proteins to DNA strand crossovers in the plasmids, in accordance with their potential to interact with both negatively and positively supercoiled DNA. In addition, the three cIF proteins were analyzed for their effects on eukaryotic DNA topoisomerases I and II. Possibly because cIF proteins interact with the same plectonemic and paranemic scDNA conformations also recognized by topoisomerases, but select the major groove of DNA for binding in contrast to topoisomerases that insert into the minor groove, the cIF proteins were able to stimulate the enzymes in their supercoil-relaxing activity on both negatively and positively supercoiled plasmids. The stimulatory effect was considerably stronger on topoisomerase I than on topoisomerase II. Moreover, cIF proteins assisted topoisomerases I and II in overwinding plasmid DNA with the formation of positive supercoils. Results obtained with the N-terminal head domain of vimentin harboring the DNA binding region and terminally truncated vimentin proteins indicated the involvement of both protein-DNA and protein-protein interactions in these activities. Based on these observations, it seems conceivable that cIF proteins participate in the control of the steady-state level of DNA superhelicity in the interphase nucleus in conjunction with such topoisomerase-controlled processes as DNA replication, transcription, recombination, maintenance of genome stability, and chromosome condensation and segregation.