PURPOSE. There is a correlation between cell volume changes and changes in the rate of aqueous humor outflow; agents that decrease trabecular meshwork (TM) cell volume increase the rate of aqueous humor outflow. This study investigated the effects of the nitric oxide (NO)-independent activators of soluble guanylate cyclase (sGC), YC-1, and BAY-58-2667 on TM cell volume and the signal transduction pathways and ion channel involved. METHODS. Cell volume was measured with the use of calcein AM fluorescent dye, detected by confocal microscopy. Inhibitors and activators of sGC, 3',5'-cyclic guanosine monophosphate (cGMP), protein kinase G (PKG), and the BKCa channel were used to characterize their involvement in the YC-1-and BAY-58-2667-induced regulation of TM cell volume. cGMP was assayed by an enzyme immunoassay. RESULTS. YC-1 (10 nM-200 μM) and BAY-58-2667 (10 nM-100 μM) each elicited a biphasic effect on TM cell volume. YC-1 (1 μM) increased TM cell volume, but higher concentrations decreased TM cell volume. Similarly, BAY-58-2667 (100 nM) increased TM cell volume, but higher concentrations decreased cell volume. The YC-1-induced cell volume decrease was mimicked by 8-Br-cGMP and abolished by the sGC inhibitor ODQ, the PKG inhibitor (RP)-8-Br-PET-cGMP-S, and the BKCa channel inhibitor IBTX. The BAY-58-2667-induced cell volume decrease was mimicked by 8-Br-cGMP and was abolished by the PKG inhibitor and the BKCa channel inhibitor. Unlike the YC-1 response, ODQ potentiated the BAY-58-2667-induced decreases in cell volume. CONCLUSIONS. These data suggest that the NO-independent decrease in TM cell volume is mediated by the sGC/cGMP/PKG pathway and involves K+ efflux.