Recent advances have been made in addressing three intriguing aspects of human genome organization: the organization of protein-coding sequences within chromosomes, the structural basis of the metaphase chromosomal banding pattern, and the function of non protein coding DNA. At the cytogenetic level, R band heterogeneity has been examined by fluorescence in situ hybridization using complex fractions of genomic DNA as probes. DNA fractionated according to GC content and CpG is island density both generated patterns related G and R bands and directly demonstrated regional variations in gene densities. A structural basis for metaphase bands has been proposed that is based on the differential size and packing of DNA loops and matrix attachment sites in G versus R bands. The model presents interesting opportunities for structure/function and organization investigations. At the molecular level, the human genome initiative has resulted in extensive genomic clone coverage for many large chromosomal regions, permitting detailed documentation of CpG islands, base composition and repeat sequence context, as well as fueling comprehensive gene searches. Sequence and functional characteristics are being examined at the kilobase level and are prompting new suggestions of roles for 'junk' DNA. Because of these developments, opportunities are now emerging for direct assessment of the molecular characteristics of individual metaphase bands.