Human astrovirus (HAstV) is the second most important cause of viral diarrhea and acute gastroenteritis in infants under five. However, determination of the infectivity of clinical isolates is difficult, and the replication cycle of HAstV is not yet fully understood. In this study, it was attempted to detect negative-sense (-)RNAs generated during the replication of RNA viruses. We used clinical isolates of HAstV to infect CaCo-2 cells. Reverse transcription using only a sense primer followed by PCR using both sense and antisense primers showed that (-)RNAs were first detected in CaCo-2 cells between 9 and 12 h postinfection (p.i.). However, these (-)RNAs were not detected when cells were treated with the protein synthesis inhibitor cycloheximide during HAstV infection. Next, RT with only an antisense primer followed by PCR was performed to detect (+)RNA of HAstVs after production of (-)RNAs during replication. RT-PCR results using the antisense primer revealed that the amount of (+)RNA began to increase starting 9 h p.i., indicating an accumulation of the newly synthesized (+)RNA genome. Cycloheximide was observed to abrogate the increase of newly made (+)RNA during HAstV infection. In conclusion, the use of sense or antisense primers during the RT reaction together with cycloheximide enabled us to quantitatively detect (-)RNAs, and this proved to be an useful tool in understanding the replication cycle of HAstV.