Semliki Forest virus (SFV) non-structural protein 1 (nsP1) is a major component of the virus replicase complex. It has previously been studied in cells infected with virus or using transient or stable expression systems. To extend these studies, tetracycline-inducible stable cell lines expressing SFV nsP1 or its palmitoylation-negative mutant (nsP16D) were constructed. The levels of protein expression and the subcellular localization of nsP1 in induced cells were similar to those in virus-infected cells. The nsP1 expressed by stable, inducible cell lines or by SFV-infected HEK293 T-REx cells was a stable protein with a half-life of approximately 5 h. In contrast to SFV infection, induction of nsP1 expression had no detectable effect on cellular transcription, translation or viability. Induction of expression of nsP1 or nsP16D interfered with multiplication of SFV, typically resulting in a 5-10-fold reduction in virus yields. This reduction was not due to a decrease in the number of infected cells, indicating that nsP1 expression does not block virus entry or initiation of replication. Expression of nsP1 interfered with virus genomic RNA synthesis and delayed accumulation of viral subgenomic RNA translation products. Expression of nsP1 with a mutation in the palmitoylation site reduced synthesis of genomic and subgenomic RNAs and their products of translation, and this effect did not resolve with time. These results are in agreement with data published previously, suggesting a role for nsP1 in genomic RNA synthesis.