We have previously introduced an optical technique for recording the transport of fluorescent substrates by single membrane transporters. Referred to as optical single-transporter recording (OSTR), the method was restricted to cases in which membrane transporters occurred at extremely small densities, namely at one or a few transporters per cell. Here we describe the extension of OSCR from whole cells harbouring a small number of transporters to small membrane patches containing transporters at normal densities. A technique was developed for firmly attaching cells to isoporous filters, i.e. very thin transparent sheets containing homogeneous populations of cylindrical pores. The flux of fluorescent transport substrates across the tiny membrane pieces spanning the filter pores was measured by scanning microphotolysis, a combination of fluorescence microphotolysis and confocal laser scanning microscopy. The technique was tested by attaching erythrocytes to filters containing pores of 1.2, 2.0 or 3.0 μm diameter. After treating filter-attached erythrocyte membranes with streptolysin O, the transport of the fluorescent protein B-phycoerythrin through single streptolysin O pores was observed. From the flux data the functional radius of the streptolysin O pore was derived to be 12.5 ± 0.9 nm, in very good agreement with previous electron microscopic estimates. The new technique features a number of unique properties: (i) the size of the membrane patch can be chosen within wide limits according to transporter density, (ii) transport can be recorded on many membrane patches in parallel, (iii) both influx and efflux may be analysed employing either photobleaching of fluorescent or photorelease of caged nonfluorescent substrates, (iv) two or more transport substrates may be monitored simultaneously. The new technique can be used, for instance, for analysing the activity of protein/particle pumps, a membrane transport domain not previously accessible to a single-transporter analysis.