Numerous combinations, concentrations, pH's and durations of HCl, NaOH and SSC treatments were tested for the purpose of developing an improved C banding technique for human metaphase chromosomes. Methods of slide preparation, as they affect C banding were also evaluated. — HCl and SSC treatment used separately, for all times and concentrations tested, gave no C banding. All treatment sequences which included an NaOH exposure gave at least some C banding, but also gave considerable swelling and distortion. Surprisingly, the best results were obtained from heat-dried preparations exposed to 0.2 N HCl at 25° C for 15 minutes, no NaOH and subsequently incubated in 2xSSC, pH=7.0 at 62–65° C for 18–24 hours. This technique is now being used routinely, following a G banding technique for homologue identification, to monitor C band variation in human chromosomes. — The pH of the 2xSSC incubation solution was found to be important. Slides treated as above with HCl, but with 2xSSC, pH=6.0 gave only G banding; HCl and 2xSSC, pH=8.0 gave C banding, but considerable chromosome swelling and poor uptake of stain. — Air- or ignition-dried preparations, with the HCl and 2xSSC treatment appeared undertreated and gave a mixture of G and C banding. A brief (30 second) exposure to 0.07 N NaOH between the HCl and 2xSSC steps is recommended. These results are in support of DNA-protein interaction and/or loss rather than denaturation-renaturation as a likely mechanism for C band production.