The rate of excision repair of UV-induced pyrimidine dimers (PDs) was measured at specific sites in each strand of a yeast minichromosome containing an active gene (URA3), a replication origin (ARS1), and positioned nucleosomes. All six PD sites analyzed in the transcribed URA3 strand were repaired more rapidly (greater than 5-fold on average) than any of the nine PD sites analyzed in the nontranscribed strand. Efficient repair also occurred in both strands of a disrupted TRP1 gene (ten PD sites), containing four unstable nucleosomes, and in a nucleosome gap at the 5' end of URA3 (two PD sites). Conversely, slow repair occurred in both strands immediately downstream of the URA3 gene (12 of 14 PD sites). This region contains the ARS1 consensus sequence, a nucleosome gap, and two stable nucleosomes. Thus, modulation of DNA repair occurs in a simple yeast minichromosome and correlates with gene expression, nucleosome stability, and (possibly) control of replication.