Deoxyribonucleic acid (DNA) fiber autoradiography is a technique that allows analysis of replication events on mammalian chromosomal DNA. 3H-thymidine is used to pulse-label DNA, which is subsequently released from lysed cells and extended linearly over a glass slide. Autoradiograms are produced by exposing the labeled DNA to light-sensitive emulsion. The effects of inhibition of protein synthesis on DNA replication were examined using this technique. Analysis of the autoradiographic patterns has shown that inhibition of protein synthesis with either cycloheximide or puromycin retards the rate of replication fork progression. In addition, these antibiotics after the normal pattern of multifocal initiation on replication units. The spatial interval between active initiation sites is decreased, there is a decay in synchrony of initiation events on subchromosomal clusters of replication units, the decay in synchrony of initiation events on subchromosomal clusters of replication units, the frequency of initiation is inhibited and the frequency of units showing the normal bidirectional mode of replication is decreased. The retarded fork progression and shorter initiation intervals may result either from the continued operation of a subset of replication units resistant to the inhibition of protein synthesis, or be manifestations of the inhibition of protein synthesis on all active sites. The other alterations, decreased synchrony and frequency of initiation and increased unidirectional replication probably occur on all active units when protein synthesis is inhibited.