We have developed a system that allows focal drug application for cell culture microscopy. Single-cell drug delivery is achieved through the insertion of a patch-clamp-like micropipette in a microenvironment-controlled chamber mounted on a standard 35-mm Petri dish. The system has precise control of temperature, CO(2) concentration, and humidity, while preventing contamination during experiments. The use of standard Petri dishes allows long-term experiments by alternating in situ microscopy with incubator growth. Modern biological long-term experiments such as the characterization of drug effects on cell movement, axonal guidance, mitosis, apoptosis, differentiation, or volume regulation can be performed. The chamber is compatible with any inverted microscope without significant modifications.