Mammalian mitochondrial genomes have a presumptive transcription termination site at the 16S rRNA-tRNALeu gene boundary. We have developed an in vitro system from human KB cells that terminates transcription at this gene boundary. By S1 nuclease protection, the 3' ends of terminated transcripts were mapped 3 and 4 base pairs upstream of the 16S rRNA-tRNALeu gene boundary, in agreement with in vivo data. By high-resolution sizing of transcripts, the 3' end was mapped 7 +/- 1 base pairs downstream from the gene boundary. Termination occurs with equal efficacy from transcriptional initiation at the heavy- or light-strand promoter. All template nucleotide sequence information necessary for termination appears to be located near the termination site itself. An unexpected observation is that the termination region functions bidirectionally.