Analysis of the retinoblastoma locus (RB1) by in situ hybridization at the infragenic level was done using human chromosomes. A high level of resolution was attained with nonisotopic in situ hybridization on banded chromosomes in both fluorescence and electron microscopy. DNA sequences less than 100 kb apart could be positioned on band 13q14 in the order expected from molecular mapping. These observations suggest that the DNA target of hybridized probes on metaphase chromosomes may correspond to nucleoprotein loops giving a resolution comparable to that obtained in interphase nuclei.