The deduced amino acid sequence of the region downstream of the reverse transcriptase (RT) motif of the Trypanosoma cruzi L1Tc non-LTR retrotransposon shows a significant homology with the sequence coding for proteins with RNase H activity from different organisms and retroelements. The 25-kDa His(6)-tagged recombinant protein bearing only the L1Tc RNase H domain, named RHL1Tc, exhibits RNase H activity as measured on the [(3)H]poly(rA)/poly(dT) hybrid used as substrate as well as on specific homologous and heterologous [(32)P]RNA/DNA hybrids. The mutation of the conserved aspartic acid at position 39 of the enzyme catalytic site, but not of the serine at position 56 (non-conservative amino acid), abolishes protein RNase H activity. The RNase H activity of the RHL1Tc protein is Mg(2+)-dependent, and it is also active in the presence of the Mn(2+) ion. The optimal condition of RNase H activity is found at pH 8 and 37 degrees C, although it also has significant enzymatic activity at 19 degrees C and pH 6. However, it cannot be excluded that the RNase H activity level and its optimal conditions may be different from that of a protein containing both RT and RNase H domains.