In the ciliary muscle, the tonic contraction requires a sustained influx of Ca²⁺ through the cell membrane. However, little has hitherto been known about the route(s) of Ca²⁺ influx in this tissue that lacks voltage-gated Ca²⁺ channels. To identify ion channels as the Ca²⁺ entry pathway we studied the effects of carbachol (CCh) on freshly isolated bovine ciliary muscle cells by whole-cell voltage clamp. Experiments were carried out using pipettes filled with K⁺-free solution containing 100 mM-Cs aspartate, 5 mM-BAPTA and 180 µM-GTP (pH 7.0; the intracellular free Ca²⁺ concentration, [Ca²⁺]_i=70 nM). CCh evoked an inward current showing polarity reversal at a holding potential near 0 mV. Analysis of the current noise distinguished two types of non-selective cation channel (NSCCL and NSCCS) with widely different unitary conductances (35 pS and 100 fS). The ratios of the permeabilities to Li⁺, Na⁺, Cs⁺, Mg²⁺, Ca²⁺, Sr²⁺ and Ba²⁺, estimated by cation replacement procedures, were 0.9 : 1.0 : 1.5 : 0.2 : 0.3 : 0.4 : 0.5 for NSCCL; and 1.0 : 1.0 : 1.8 : 2.5 : 2.6 : 3.2 : 5.0 for NSCCS. NSCCS, but not NSCCL, was strongly inhibited by elevation of [Ca²⁺]_i. Both NSCCL and NSCCS were dose-dependently inhibited by 1-100 µM of SKF96365, La³⁺ and Gd³⁺, which also inhibited the tonic component of the contraction produced in muscle bundles by CCh without markedly affecting the initial phasic component. NSCCL and/or NSCCS may serve as a major Ca²⁺ entry pathway required for sustained contraction of the bovine ciliary muscle. RT-PCR experiments in the bovine ciliary muscle (whole tissue) detected mRNA's of several transient receptor potential (TRP) channel homologues (TRPC1, TRPC3, TRPC4 and TRPC6), which are now regarded as possible molecular candidates for receptor-operated cation channels.