Artificial?chromosomes were previously generated for use in bacteria, protists, yeast and human cells. A?Tetrahymena?thermophila?artificialchromosome?could serve as a versatile platform to study diverse aspects of?Tetrahymena?biology and beyond. Here, we placed a C3-type rDNA replication origin and telomere sequences from T.?thermophila?into a pNeo4 vector, producing the first T.?thermophila?macronuclearartificial?chromosome?(TtAC1). Circular or linear forms of TtAC1 can be stably transformed into both vegetative and conjugative T.?thermophila?cells. Linear TtAC1 was stably double in copy number under antibiotic selection, but its copy number was dropping without antibiotic selection pressure. Southern blot, Real-Time PCR and E. coli retransformation analyses together showed that TtAC1 vector did not integrate into the?macronuclear?genome, and was maintained as a linear or a circular?chromosome?in T.?thermophila?macronucleus under antibiotic selection. The use of TtAC1 for recombinant protein production was demonstrated by western blot analysis of a secreted 27 kDa TtsfGFP-12XHis protein. We present the first?macronuclear?artificial?chromosome?with species-specific chromosomal elements for use in T.?thermophila?studies and to aid broad recombinant biotechnology applications.