The mRNA for human thyroid peroxidase (hTPO) is 3.1 kilobases (kb) in size, coding for a protein of 933 amino acids. However, there is controversy as to whether other hTPO mRNA transcripts exist in thyroid cells. There is one report of the existence of 2.1- and 1.7-kb transcripts (hTPO mRNA species I and II), representing up to half of the hTPO mRNA in TSH-stimulated human thyroid cells. On the other hand, numerous other studies have only observed 3.1-kb hTPO mRNA transcripts. The nature of these putative 2.1- and 1.7-kb mRNA transcripts, if present, is unknown. We now report the isolation and characterization of two smaller hTPO cDNA species, designated hTPO cDNA I and II. cDNA I and II are 1753 and 1044 basepairs (bp) in size, with open reading frames of only 225 and 174 amino acids, respectively. Comparison of the nucleotide sequences of cDNA I and II with available hTPO genomic sequence reveals that cDNA I consists of exons 1-6 (654 bp) and the 5'-end of intron 6 (1099 bp); cDNA II contains exons 1-5 (486 bp) and an unidentified DNA tract of 558 bp further down-stream, presumably an intron. Confirmation that cDNA I and II correspond to mRNA transcripts I and II, respectively, was provided by Northern blot analysis with DNA probes specific for cDNA I and II. hTPO mRNA transcripts I and II are present in TSH-stimulated and TSH-deprived human thyroid cells in culture as well as in intact thyroid tissue. In summary, the present data 1) demonstrate directly that hTPO mRNA transcripts I and II exist in human thyroid cells, 2) explain the discrepant data in the literature regarding their existence, 3) elucidate their molecular structure, 4) indicate that they are generated by alternative splicing, and 5) demonstrate that hTPO mRNA I and II exist in the thyroid gland in vivo as well as in thyroid cells in culture.