Differential DNA replication is widely held to influence polytene chromosome structure by causing the dramatic reductions in heterochromatic DNA content that are characteristic of most endopolyploid cells. The "underreplication model" of heterochromatic sequence underrepresentation predicts that replication intermediates should populate regions of DNA between fully polytenized euchromatic sequences and underpolytenized heterochromatic sequences. We directly tested this prediction using Dp1187, a 1300 kb Drosophila minichromosome containing well-defined heterochromatic regions. DNA from a euchromatic/heterochromatic junction region of Dp1187, demonstrating a significant gradient of underrepresentation in larval salivary glands, lacked the stalled replication forks predicted by the underreplication model. We consider an alternative mechanism leading to heterochromatic sequence underrepresentation involving a process of DNA elimination.