The Kluyveromyces lactis HIS4 gene (KlHIS4) is transcriptionally regulated by the carbon source. The promoter region encompassing positions -238 to -139 is responsible for this regulation according to lacZ reporter assays. Electrophoretic Mobility Shift Assay (EMSA) experiments on KlHIS4 promoter (positions -218 to -213, Fragment 6, F6) show a specific gel-shift band, CS1, whose intensity is carbon-source dependent in K. lactis hap2 (klhap2) knock-out strains. The klhap3 mutation is not able to cause this effect by itself, but the combination of klhap2 and klhap3 mutations has an enhanced effect on CS1 band formation. Introducing a heat shock element (HSE) at the sequence in the F6 fragment (mutated F6, F6*) increases the binding activity in the klhap2 mutant. KlHIS4 mRNA levels in the klhap2 or the double Klhap2/3p mutant do not correlate with the increase in CS1 binding activity, indicating that the factor causing CS1 is acting and only detectable in vitro. EMSA experiments with K. lactis wild-type cells under temperature stress conditions show a band enhancement (Ts1), similar in size to CS1. Cross-competition experiments between F6 and F6* show that F6* competes more efficiently than F6 for both CS1 and Ts1 formation, indicating the involvement of the HSE in the formation of the specific gel-shift bands. Moreover, the similar gel-shift patterns suggest that both bands are caused by the same heat shock-like factor under different stress conditions. Therefore, the enhancement of the CS1 band signal in the klhap2 (and klhap2/3) mutants is due to the increase in heat shock-like factors in the protein extracts from these mutant cells grown in a non-fermentable carbon source. This Klhap2-dependent stress effect was not previously described in K. lactis.