We detected an unexpected small-sized DNA fragment during polymerase chain reaction (PCR) analysis of the heterogeneity of a macronuclear intergenic region of Paramecium caudatum. Southern blotting of total genomic DNA with the PCR product as a probe indicated that the small-sized DNA fragment constituted part of the macronuclear genome. Sequencing revealed that the PCR product was a chimeric DNA structure that may be generated by tail-to-tail fusion of the 5' region of the hemoglobin (hb) gene to most of the nucleosome assembly protein-1 (nap-1) gene. Short tandem repeats consisting of tetra- and tri-nucleotides exist at the putative cleavage sites in the hb and nap-1 genes, respectively. This feature differs from those found at the boundaries of TA-internal eliminated sequences in the P. aurelia complex and at transposable elements in other species. This suggests that the chimeric DNA is generated by a novel mechanism. Although the chimeric DNA contains the hb and nap-1 promoters, transcripts corresponding to the chimeric DNA were not detected by reverse transcription (RT)-PCR analysis during vegetative cell growth. Possible roles of chimeric DNA are discussed.