The Miller spreading procedure was applied to mouse metaphase spreads of methotrexate-resistant 3T3 cells that contain large numbers of minute chromosomes and dihydrofolate reductase genes. There is substantial variation in both size and numbers of minutes in individual cells, the smallest of which (estimated as 5 X 10(3) kilobase pairs) would be undetected by standard light microscopic analyses. Minute chromosomes are composed of nucleosomal chromatin, which is organized into typical higher order fibers that are folded to form rosette-like structures characteristic of normal chromosome organization. There is no evidence that the DNA in minutes is linear. Minutes exist singly and in pairs, and members of a pair are connected by higher order chromatin fibers, suggesting that they are topologically interlocked. They are often closely apposed to chromosomal telomeres or arms, a configuration that may be involved in their distribution at mitosis. In addition to typical minutes, which do not possess kinetochores, a small marker chromosome possessing all of the features of a centromere region is present in parental and resistant cells. An unusual feature of this cell line is the retention of resistance, minute chromosomes, and amplified dihydrofolate reductase genes; most methotrexate-resistant mouse cell lines with minute chromosomes lose these properties when grown in the absence of methotrexate.