Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus that causes severe persistent arthralgia. To better understand the molecular details of CHIKV RNA synthesis and the mode of action of inhibitors, we have developed an in vitro assay to study CHIKV replication/transcription complexes isolated from infected cells. In this assay (32)P-CTP was incorporated into the CHIKV genome, subgenomic (sg) RNA and into a ~7.5 kb positive-stranded RNA, termed RNA II. We mapped RNA II, which was also found in CHIKV-infected cells, to the 5' end of the genome up to the start of the sgRNA promoter region. Most of the RNA-synthesizing activity, negative-stranded RNA and a relatively large proportion of nsP1 and nsP4 were recovered from a crude membrane fraction obtained by pelleting at 15,000 G: . Positive-stranded RNA was mainly found in the cytosolic S15 fraction, suggesting it was released from the membrane-associated replication/transcription complexes (RTCs). The newly synthesized RNA was relatively stable and remained protected from cellular nucleases, possibly by encapsidation. A set of compounds that inhibit CHIKV replication in cell culture was tested in the in vitro RTC assay. In contrast to 3'dNTPs, chain terminators that acted as potent inhibitors of RTC activity, ribavirin triphosphate and 6-aza-UTP did not affect the RNA-synthesizing activity in vitro. In conclusion, this in vitro assay for CHIKV RNA synthesis is a useful tool for mechanistic studies on the RTC and mode of action studies on compounds with anti-CHIKV activity.