Little is known about the mechanisms by which Ca²⁺-binding sensory proteins direct the plant heat shock (HS) response. Since two Ca²⁺-dependent protein kinases (CPK3 and CPK13) were recently shown to phosphorylate the heat shock transcription factor HsfB2a, we assessed in the current study whether these kinases are also involved in HS signal transduction, by monitoring the transcriptional profile of HS protein (Hsp) family genes in Arabidopsis Col-0 plants (WT) and the corresponding mutants. Both with and without HS, the gene transcript levels of Hsp70, Hsp101, Hsp17.4-CIII and Hsp15.7-CI were found to be lower in cpk3 and cpk13 mutants compared to WT, resulting in the impairment of basal thermotolerance in the mutants. To determine the in vivo function of CPKs, CPK3/13 and their substrate HsfB2a (heat shock transcription factor) were co-expressed as cofactors for the transient expression of a reporter (GUS) gene under the control of heat shock element (HSE) in Nicotiana benthamiana leaves. However, CPK3/13-phosphorylated HsfB2a did not function in the suppression/activation of HSE-promoted expression in the transient expression system. Implications for possible signal trafficking via CPKs and Hsfs are discussed.