Iron is an indispensable micronutrient for virtually all microorganisms, where it acts as a cofactor of many enzymes involved in regulation of multiple cellular and physiological functions. This metal is also considered an important determinant contributing to the pathogenesis of fungal infectious diseases, and therefore the identification of iron-regulated metabolic processes occurring within the invading fungal cell can help the development of new antifungal therapeutic strategies. In this study, we examined relationships between iron availability and neutral storage lipids in Histoplasma capsulatum, a dimorphic fungus responsible for the most common respiratory and systemic mycosis in humans. Yeast cells were grown in a defined minimal medium supplemented with or without iron. Lipids were extracted from cells at the log and late stationary growth phases, then separated by thin-layer chromatography, and fatty acids were analyzed by gas chromatography. A culture age-related decrease in the unsaturated fatty acid content was observed in all four neutral lipid classes examined. Iron-related alterations could be seen in relation to triacylglycerol and free fatty acid pools, whereas no iron-dependent effects were detected in diacylglycerol and steryl ester fractions. Regarding triacylglycerols, the presence of iron positively affected the content of unsaturated fatty acids, and this stabilizing action of iron was notably increased when ferrous ions were added. Subsequent iron uptake studies showed a definite preference of H. capsulatum to acquire iron in its reduced, more soluble, ferrous form, and therefore, the availability of iron may be the underlying reason for the observed iron-maintained homeostasis in H. capsulatum triacylglycerols.