Although the existence of a cellular heat shock response is nearly universal, its relationship to organismal thermal tolerance is not completely understood. Many of the genes involved are known to be regulated by the highly conserved heat shock transcription factor-1 (HSF-1), yet the regulatory network is not fully characterized. Here, we investigated the role of HSF-1 in gene expression following thermal stress using knockdown of HSF-1 by RNA interference in the intertidal copepod Tigriopus californicus We observed some evidence for decreased transcription of heat shock protein genes following knockdown, supporting the widely acknowledged role of HSF-1 in the heat shock response. However, the majority of differentially expressed genes between the control and HSF-1 knockdown groups were upregulated, suggesting that HSF-1 normally functions to repress their expression. Differential expression observed in genes related to chitin and cuticle formation lends support to previous findings that these processes are highly regulated following heat stress. We performed a genome scan and identified a set of 396 genes associated with canonical heat shock elements. RNA-seq data did not find those genes to be more highly represented in our HSF-1 knockdown treatment, indicating that requirements for binding and interaction of HSF-1 with a given gene are not simply predicted by the presence of HSF-1 binding sites. Further study of the pathways implicated by these results and future comparisons among populations of T. californicus may help us understand the role and importance of HSF-1 in the heat shock response and, more broadly, in organismal thermal tolerance.,? 2020. Published by The Company of Biologists Ltd.