Analysis of 3H-thymidine autoradiograms of late third instar larval salivary glands of Drosophila pseudoobscura revealed a unique example of asynchrony of replication in the autosome complement. The two autosomal arms, 2 and 3, show similar labeling pattern during the initial phases, DD to 3C, and thereafter, the chromosome 3 has fewer labeled sites than chromosome 2 until the most terminal pattern, 1D. Detailed sitewise analysis of 3H-thymidine labeling shows that while nearly 54% of the sites examined in chromosome 2 have a labeling frequency greater than 50%, only 13% of all sites in chromosome 3 have labeling frequency at that range. The number of labeled sites on chromosome 3 plotted against that on chromosome 2 shows a hyperbolic profile rather than a linear relationship. The silver grain ratio of the 2nd to 3rd increases from 1.5 to 3.1 through different stages of the cycle. These results suggest that both chromosomes start replication simultaneously but the third chromosome appears to complete the replication earlier than the second. These data open up the possibility of separate control mechanisms for the initiation and termination of DNA replication in polytene chromosomes.