A hallmark of human cytomegalovirus (HCMV) infection is the characteristic enlargement of the host cells (cytomegaly). Since iron (Fe) is required for cell growth and Fe chelators will inhibit viral replication, we investigated the effects of HCMV infection on Fe homeostasis in MRC-5 fibroblasts. Using the metallo-sensitive fluorophore calcein and the Fe chelator salicylaldehyde isonicotinoyl hydrazone (SIH), the labile iron pool (LIP) in mock-infected cells was determined to be 1.04±0.05 µM. Twenty-four hours post infection (h.p.i.), the size of the LIP was nearly doubled. Since cytomegaly occurs between 24 and 96 h.p.i., access to this larger LIP could be expected to facilitate enlargement to ~375% of the initial cell size. The ability of Fe chelation (SIH, 100 µM) to limit enlargement to ~180%, confirms that the LIP plays a major role in cytomegaly. The effect of SIH chelation on the mitochondrial membrane potential (_M) and morphology was studied using the mitochondrial voltage-sensitive dye, JC-1. The mitochondria in mock-infected cells were heterogeneous with a broad distribution of _M and thread-like. In contrast, the mitochondria of HCMV-infected cells had a more depolarized _M distributed over a narrow range and were grain-like in appearance. However, the HCMV-induced alteration in _M was not affected by SIH chelation. In conclusion, the development of cytomegaly is inhibited by Fe chelation and may be facilitated by an HCMV-induced increase in the LIP.