Responses of the bovine iris sphincter and dilator muscles evoked either by single electrical stimulation (1–50 ms) or repetitive pulses (0.1–1 ms for 0.1–0.7 s) were isometrically recorded in vitro. In response to electrical nerve stimulation, the bovine iris sphincter exhibited cholinergic contraction followed by slow relaxation, while the dilator predominantly showed cholinergic relaxation. In order to compare the time courses of the iris sphincter and the dilator, the two muscle strips were investigated under the same conditions. Regardless of the conditions of electrical stimulation and incubation time, significant differences were found between the iris sphincter and the dilator in their response time courses.