Four individuals carrying different Robertsonian translocations (13q;14q, 14q;21q, 14q;15q, and 13q;21q) were studied to determine the breakpoints involved in the generation of these derivative chromosomes. Sequential high-resolution G-banding, in situ hybridization using alphoid and ribosomal DNA probes, and C-banding were performed. In addition, silver staining was also used for visualization of the NOR region. The results provide direct molecular cytogenetic evidence that Robertsonian translocations can take place in different regions in both the short arm and proximal long arm of acrocentric chromosomes. Three different types of breakpoints were identified: between the ribosomal or alphoid sequences, as deduced from the banding and in situ hybridization results, and breaks in two seemingly unrelated regions on the two different chromosomes. The use of conventional cytogenetic techniques together with molecular studies allowed more precise evaluation of the breakpoints involved in Robertsonian translocations than either approach alone might have done.