We present results from a nonautoradiographic study of DNA replication in polytene chromosomes from dipteran larvae. Monoclonal antibodies with specificity for 5-bromodeoxyuridine (BrdUrd) were used to localize by indirect immunofluorescence the sites of BrdUrd incorporation and to follow the dynamics of DNA synthesis in salivary gland cells of 4th instar Chironomus thummi larvae.This technique presents numerous advantages over autoradiographic procedures and allows mapping of DNA synthesis patterns at the level of resolution of one chromosomal band. Several replication patterns were observed, classified according to characteristic features, and tentatively assigned to specific periods of the S-phase. In early S-phase, DNA synthesis is first detectable in puffs and interbands. later in bands. Most chromosomal bands appear to initiate DNA synthesis synchronously; however, in bands within centromeric and heterochromatic regions the start of synthesis is delayed. At mid S-phase, all the bands show uniform staining. Subsequent staining patterns are increasingly differential with the bands displaying characteristic fluorescence intensities. As replication progresses through the late S-phase period, the chromosomes show a decreasing number of fluorescent bands. The last bands to terminate replication are located in centromeric and heterochromatic DNA-rich regions and a few bands of low DNA content in region IIAa-c.Key terms: DNA replication, 5-bromodeoxyuridine, monoclonal anti-BrdUrd antibodies, polytene chromosomes Successive endoreduplication cycles during the larval growth of dipteran insects give rise to the formation in certain tissues of giant polytenic chromosomes (3,23). In the salivary gland cells of Chironomus species, 12 to 13 rounds of DNA replication during embryonic and larval development (15) result in the formation of polytene chromosomes with up to Z13 chromatids. Due to their size, the constancy of their band-interband pattern, the equation of bands with genes, and the structural changes that accompany gene activation (puffing), these chromosomes present a unique opportunity for studying in situ the structural organization of active and inactive chromatin, the distribution of chromosomal proteins and of particular types of DNA sequences, the mechanisms of gene activation, and the dynamics of DNA replication (3,251.DNA replication in polytene chromosomes of dipteran larvae has been the subject of numerous studies (1,2,4,5,8,17,20,21), which have made use of autoradiographic techniques to detect the incorporation of 3H-thymidine. Various patterns of DNA synthesis have been described and tentatively ordered on a time scale. Patterns characteristic of early S-phase display more or less continuous labeling, while late S-phase patterns are of a discontinuous type. In Drosophila, a discontinuous pattern with label confined to puffs and interbands was observed by Hagele and Kalisch (10,19) and assigned to the very beginning of the replication phase. It was concluded from these experiments that the various ...