Electron microscopy has been used to study the fine structural organization of R-banded chromosomes prepared by treatment of the chromosomes with a hot NaH2PO4 solution. The results indicate that there is a structural basis for R-banding with this technique. In comparison to untreated control chromosomes, the R-banded chromosomes had a greatly reduced electron density, suggesting that the heat treatment has a general adverse effect on chromosome structure. Chromatin fibers formed a coarse, irregular network throughout the chromosome and were often enlarged, probably as a result of the fusion of two or more native fibers. The chromatin fibers were more aggregated and had an increased electron density in the R-band regions of the chromosome than in the interbands. This indicates that the treatment has a differential effect on the structure of bands and interbands. A comparison of the ultrastructure of R- and G-banded chromosomes demonstrated that the distribution of aggregated chromatin was reversed by these two types of banding techniques; however, the treatments producing the R-banding appeared to induce less extreme differences in the degree of chromatin condensation in band and interband regions than those giving rise to G-banding. It is suggested that alterations of DNA-protein interactions may arise from the differential denaturation of proteins and/or DNA in R-band and interband regions during the heat pretreatment. Such differential alterations in DNA-protein interactions may induce localized changes in the organization of chromatin and may account for the subtle morphological differences observed between the band and interband regions.