Macronuclear chromosomes of spirotrichous ciliates are mainly "nanochromosomes" containing only a single gene. We identified a two-gene chromosome in the spirotrich Sterkiella histriomuscorum (formerly Oxytricha trifallax) which, unlike other characterized two-gene molecules, contains reading frames oriented tail to tail. These are homologs of ribosomal protein L29 (RPL29) and cyclophilin. We found that both genes are transcribed, with their polyadenylation sites on opposite strands separated by only 135 bp. Furthermore, both genes in S. histriomuscorum are present only on one macronuclear chromosome and do not occur alone or linked to other genes. The corresponding micronuclear locus is fragmented into three nonscrambled gene segments (MDSs), separated by two noncoding segments (IESs). We also found that these two genes are linked on a macronuclear chromosome, similarly arranged tail to tail, in the three spirotrichs Stylonychia lemnae, Uroleptus sp., and Holosticha sp.. In addition, single-gene macronuclear chromosomes containing only the RPL29 gene were detected in the earlier diverged Holosticha and Uroleptus. These observations suggest a possible evolutionary trend towards loss of chromosomal breakage between these two genes. This study is the first to examine gene linkage in the macronucleus of several spirotrichs and may provide insight into the evolution of multi-gene macronuclear chromosomes and chromosomal fragmentation in spirotrichs.