The pharmacological effects of glargine, protamine zinc (PZI), and lente insulins were evaluated in nine healthy cats. A 3-way crossover study was performed and plasma concentrations of insulin and glucose were determined for 24 h after a single subcutaneous injection of each insulin at 3-day intervals. Time to onset of action did not differ between insulins. Mean time to first nadir glucose was longer for glargine (14 h) relative to PZI (4 h) and lente (5 h). PZI was biphasic in action with nadirs at 4 and 14 h with the second nadir occurring at a similar time to glargine. Nadir glucose did not differ significantly between insulin types. The duration of action was similar for glargine and PZI and was longer than that for lente insulin. Mean daily glucose after glargine and PZI were also similar and were lower than after lente insulin. Time to reach peak insulin did not differ between insulin types. Time to return to baseline insulin level for PZI was longer than glargine but did not differ significantly from lente. In conclusion, healthy cats injected subcutaneously with glargine, compared to those injected with lente insulin, have a later glucose nadir and longer duration of action. Glargine and PZI had similar durations of action in study cats but a larger study is required to obtain precise comparisons of duration of action.