During a hospital epidemic of infections with gentamicin-resistant Serratia marcescens (GRS), we studied the relation between receiving antibiotics and acquiring GRS. In a five-month period, 22 patients acquired GRS, whereas 18 patients acquired gentamicin-sensitive Serratia (GSS). When compared with patients with nosocomial GSS infection, patients with nosocomial GRS had been in the hospital (p = 0.04) and the intensive care unit (p = 0.003) longer before infection and more had received gentamicin (p = 0.001) or ampicillin (p = 0.02) before infection. To control for the influence of underlying disease, we matched all 12 ICU patients with GRS infection and 12 patients without GRS infection for underlying illness and duration of intensive care. Use of any antibiotic (p = 0.04), or a combination of gentamicin plus ampicillin or cephalosporin (p = 0.047) was more common among patients with GRS infection. The hospital had not significantly increased the use of aminoglycosides from the previous year. We conclude that for the individual patient antimicrobial therapy, especially with gentamicin or ampicillin, creates a risk for later infection by GRS that is independent of the severity of the underlying illness.