We report the characterization of a transcription unit at the second chromosome locus 36B, designated TU-36B which is adjacent to the 3' end of the Drosophila myosin heavy-chain (Mhc) gene. We have isolated and sequenced a complementary DNA clone and the region of genomic DNA which represents this gene. The sequencing studies reveal that this gene contains one intron, the mRNA is 1480 nucleotides in length, the TU-36B mRNA is transcribed in an orientation opposite to the Mhc mRNAs, and the poly(A) addition site of this gene is 99 nucleotides downstream of poly(A) addition site A-2 of the Drosophila Mhc gene. The mRNA contains a continuous open reading frame which can encode a protein product of 47,000 daltons in molecular weight. The proposed protein shares homology with cytochrome b proteins. Comparison of in situ hybridization of Mhc specific and TU-36B specific probes to tissue sections demonstrates that both mRNAs are predominantly transcribed in the same muscle tissues of the developing fly.