Many sequences in eukaryotic genomes have the potential to adopt a left-handed Z-DNA conformation. We used a previously described assay based on the binding of a mAb to Z-DNA to inquire whether Z-DNA is formed in the rat nucleolin ( Ncl ) gene in metabolically active, permeabilized nuclei. Using real-time PCR to measure Z-DNA formation, the potential Z-DNA sequence element Z1 [(CA) ₁₀ (CG) ₈ ] in the promoter region was found to be enriched 571- to 4,040-fold in different cell lines, whereas Z2 [AC(GC) ₅ CCGT(CG) ₂ ] in the first intron was enriched 12- to 34-fold. Ncl promoter activity was 1.5- to 16-fold stronger than that of the simian virus 40 promoter and enhancer. This activity was further increased 36–54% when Z1 was deleted. The inhibitory effect of Z1 on Ncl promoter activity was independent of location and orientation. The Ncl Z1 element is identical to the genetic marker D9Arb5. Five allelic variants of Z1 were identified by sequence analysis of genomic DNA from various rats. The two most common alleles differed significantly (up to 27%) in their capacity to inhibit Ncl promoter activity. This finding suggests that differences in Z-DNA formation by polymorphic dinucleotide repeats may be one of the factors contributing to genetic variation.