Human cathepsin L (hCATL) has been implicated in a variety of physiological and pathological processes. It was hitherto known to be encoded by four mRNA species, namely hCATL A, AI, AII and hCATL B, differing in their 5' untranslated regions (UTRs). Of these, hCATL A, AI and AII are produced by the alternative splicing of the same primary transcript. HCATL AI and hCATL AII, lack 27 and 90 bases, respectively, from the 3' end of exon 1 of hCATL A. The present study describes the identification of a new splice variant hCATL AIII, which similarly lacks 145 bases from the 3' end of exon 1 of hCATL A. It is produced by the splicing out of 136-280 bases of the first exon in addition to intron 1 of hCATL A, which together serve as an intron for hCATL AIII. HCATL AIII was observed to be the most abundant splice variant in five different human cell lines. In vitro transcription coupled translation studies revealed that hCATL AIII is translated with 4.4-, 3.9- and 1.6-fold higher efficiency as compared to hCATL A, AI and AII, respectively. These results were further confirmed by measuring the enzymatic activities of the in vitro translated products. Cloning of hCATL AIII UTR upstream to luciferase reporter gene resulted in a 3.75-fold higher expression of the reporter gene as compared to the luciferase construct containing UTR of hCATL A. Thus, we have identified a novel human cathepsin L splice variant, hCATL AIII, which is most abundant in human cell lines and is translated with highest efficiency. Our results demonstrate either the presence of a positive or absence of a negative cis-acting regulatory element(s) in the UTR of hCATL AIII that is sufficient to confer translational advantage to a heterologous mRNA. The predominance of this most efficiently translated splice variant in malignant cells suggests that it plays a key role in the over-expression of human cathepsin L in cancer.