Since the development of the enhancer trap technique, many large libraries of nuclear localized lacZ P-element stocks have been generated. These lines can lend themselves to the molecular and biological characterization of new genes. However they are not as useful for the study of development of cellular morphologies. With the advent of the GAL4 expression system, enhancer traps have a far greater potential for utility in biological studies. Yet generation of GAL4 lines by standard random mobilization has been reported to have a low efficiency. To avoid this problem we have employed targeted transposition to generate glial-specific GAL4 lines for the study of glial cellular development. Targeted transposition is the precise exchange of one P element for another. We report the successful and complete replacement of two glial enhancer trap P[lacZ, ry+] elements with the P[GAL4, w+] element. The frequencies of transposition to the target loci were 1.3% and 0.4%. We have thus found it more efficient to generate GAL4 lines from preexisting P-element lines than to obtain tissue-specific expression of GAL4 by random P-element mobilization. It is likely that similar screens can be performed to convert many other P-element lines to the GAL4 system.