OBJECTIVE The series of experiments described were undertaken to evaluate the use of a cell-free lysis model to measure membrane fusion events in photoreceptor rod outer segments (ROS). The experiments measure fusion initiated with the osmotic disruption of fluorescenty-labeled ROS and correlate these findings with previously described disc-plasma membrane fusion. The influence of calcium and disc membrane cholesterol content on fusion between ROS membrane species was evaluated. METHODS Membrane fusion was followed by measuring the dilution of a membrane-associated fluorophore (R18) from labeled plasma membrane to an unlabeled membrane species; discs. Free calcium in the ROS preparations was measured using the calcium-sensitive fluorophore Quin-2. The affects of cholesterol content on fusion were investigated by increasing and decreasing disc membrane cholesterol content using well established lipid exchange techniques. RESULTS There is an increase in R18 fluorescence on hypotonic lysis of ROS whose plasma membrane is labeled with R18. This increase in fluorescence is inhibited by EGTA and requires nanomolar levels of calcium. The initial rates of fusion between R18-labeled plasma membrane and discs were virtually identical in discs with increased and decreased levels of membrane cholesterol. CONCLUSIONS The increase in R18 fluorescence on lysis of R18-labeled ROS is consistent with fusion between disc membranes and the surrounding plasma membrane. This fusion is dependent on nanomolar levels of calcium, is inhibited by EGTA, and is independent of the cholesterol content of these membranes.