Previous studies have shown that the differential regulation of mouse somatic cytochrome c (cyt cS) and testicular cytochrome c (cyt cT) during spermatogenesis is accompanied by changes in mRNA length [Hake et al. (1990) Development, 110, 249-257]. When analyzed by polysomal gradient sedimentation, cytochrome cT sediments in two broad size classes: non-polysomal mRNAs are about 0.6 to 0.75 kb and polysomal mRNAs range from 0.7 to 0.9 kb. Both classes of mRNAs shorten to about 0.5 kb following deadenylation. Oligonucleotide-directed cleavage of the cytochrome cT RNAs by RNase H reveals that the size heterogeneity of cytochrome cT mRNAs resides in the 5' untranslated regions (UTRs). Ribonuclease protection assays reveal that multiple cytochrome cT mRNAs are transcribed from six different transcriptional start sites spanning a region of 59 nucleotides in the 5'UTR from +1 to +59. Transcripts derived from the first and second transcriptional initiation sites are not loaded onto polysomes as efficiently as those transcripts initiated from the other start sites. Each of the longer mRNAs has an upstream open reading frame, which starts at +8 and ends at +136 in the 5'UTR of the cytochrome cT transcript. Computer analysis suggests that the lengthened 5'UTR sequences allow additional hairpin structures to be formed. Since the upstream open reading frame and the additional stem loop structure are absent in the 5' UTRs of the cytochrome cT mRNAs initiated from the four downstream start sites, we suggest that these sequences in the two longest cytochrome cT transcripts hinder their loading onto polysomes.