The alternative RNA processing pathways in human calcitonin gene (CALC-I gene) expression were investigated using steady state RNA isolated from human medullary thyroid carcinoma (MTC) and from a culture line derived from this tumor. On Northern blots the mature 1.0 kilobases (Kb) calcitonin (CT) - and 1.1 Kb calcitonin gene-related peptide (CGRP) mRNAs were detected with CALCI gene specific probes as well as high molecular weight poly (A) containing RNAs of 2.1, 2.3, 3.3, 4.2, 5.0 and 5.7 Kb. The 5.7 Kb RNA was identified as the poly(A) tailed primary transcript containing sequences corresponding to all 6 exons and 5 introns of the CALC-I gene. From the composition of the other RNAs the splicing order of the different introns could be deduced. The results suggest the following model. First all introns not involved in alternative processing (introns 1, 2 and 5) are spliced from the 5.7 Kb RNA in rapid successive reactions yielding a 3.3 Kb RNA, which accumulates. From this 3.3 Kb RNA, the last common intermediate in the alternative processing pathway, CT mRNA is formed by splicing of intron 3 and poly(A) addition at exon 4, in this order or the reverse order via 2.3 Kb or 2.1 Kb RNA intermediates respectively. Alternatively, the whole intron 3-exon 4-intron 4 region is spliced from the 3.3 Kb RNA yielding CGRP mRNA. The temporal sequence of poly(A) addition at exons 4 and 6 may relate to the observed structural differences between the poly(A) addition signals at these sites. The ratio of CT- to CGRP mRNA may relate also to the differences in the primary structures of the intron 3- and intron 4 splice acceptor sites.