We have isolated and characterized the Δ⁹-desaturase gene (Ole1), which codes for a key enzyme involved in regulating membrane fluidity in animal cells and microorganisms, from two strains ofHistoplasma capsulatum, one that is temperature-tolerant (G217B) and the other temperature-susceptible (downs). These pathogenic fungi are dimorphic in that they undergo a morphologic transition from the mycelial to yeast-like form when the temperature of incubation is switched from 25 to 37°C or when they infect a susceptible host. The coding sequences of the two genes, both containing an intron of 93 nucleotides, arevirtually identical and analogous to the Δ⁹-desaturase gene ofSaccharomyces cerevisiae and those of the rat, mouse and human.Ole1 transcription of the thermotolerant G217B and thermosensitive Downs strains is similar in yeast phase cells and during the temperature shift down from 34, 37, or 40 to 25°C (yeast-to-mycelia transition). Nevertheless, Δ⁹-desaturase gene is transcriptionally inactive in mycelia of G217B at 25°C while it is actively transcribed in the Downs strain at the same temperature. These results are in agreement with the finding that membranes of the Downs strain have a higher level of oleic acid. The differential expression of Δ⁹-desaturase genes is discussed in relationship to differences in thermosensitivity in the fungal isolates and in regulating the level of expression of heat shock genes.