Infection with the Epstein-Barr virus (EBV) sometime after childhood is the major cause of mono. However, infection with a similar virus called cytomegalovirus may cause a mono-like syndrome.

The Epstein-Barr virus infects cells of the immune system called B lymphocytes. The infection causes an increase in the numbers of white blood cells called mononuclear leukocytes, which gives the disease its name (mononucleosis). The infection causes an increase in size of the immune system organs that normally house these cells including the spleen, tonsils, and lymph nodes.

After the initial infection with EBV, there is an incubation period of four to six weeks, during which time the virus is transmissible to others even thought the person is not showing any symptoms. After symptoms appear, the immune system eventually gets the upper hand on the virus and symptoms usually disappear after a month or two. Even after symptoms are gone, the virus can still be passed to others for quite some time. Since the virus never goes away completely, it may be possible for a previously infected person to infect another person (with whom they share prolonged close contact) at any time for the rest of their life.