The aim of this one-group, quasi-experimental study was to examine the effect of choice of injection site and injection duration on the intensity of pain associated with intramuscular penicillin injection. Injections containing the same dose of drug were administered 12 hours apart for each patient over 5 s/mL and 10 s/mL durations in the dorsogluteal and ventrogluteal sites. Sixty patients who had a medical order for intramuscular penicillin at least twice in a day and for two successive days at the same dose were included in the study. No difference in pain was perceived by participants between the two injection durations at either the dorsogluteal or the ventrogluteal site. This study showed that intramuscular penicillin can be administered to either site over 5 s/mL or 10 s/mL durations. There is a need for further research with a randomized controlled design in different settings and in a larger sample on the impact of choice of injection site and injection duration on pain intensity.