We present a new method for the construction of subtracted cDNA libraries. It makes use of two directional cDNA libraries in phagemid vectors and has two advantages over traditional protocols: it can be used when a limiting amount of driver material is available and the cDNA inserts in the subtracted libraries are long enough to encode functional proteins. Using this method, we have constructed two subtracted libraries enriched for sequences expressed in the mesoderm of early Xenopus gastrulae.