Gibberellins (GAs) are phytohormones required for normal growth and development in higher plants. The Dwarf3 (D3) gene of maize encodes an early step in the GA biosynthesis pathway. We transposon-tagged the D3 gene using Robertson's Mutator (Mu) and showed that the mutant allele d3.2::Mu8 is linked to a Mu8 element. The DNA flanking the Mu8 element was cloned and shown to be linked to the d3 locus by mapping in a high-resolution population developed by selecting for recombination between d3 and linked genetic markers. To establish unambiguously the identity of the cloned gene as D3, a second mutant allele of D3 (d3.4) was also cloned and characterized using the d3.2::Mu8 sequences as a probe. d3.4 was found to have a novel insertion element, named Sleepy, inserted into an exon. A third mutant allele, d3.1, which has the same size 3' restriction fragments as d3.4 but different 5' restriction fragments, was found to contain a Sleepy insertion at the same position as d3.4. On the basis of the pedigree, Sleepy insertion, and restriction map, d3.1 appears to represent a recombinational derivative of d3.4. The D3 gene encodes a predicted protein with significant sequence similarity to cytochrome P450 enzymes. Analysis of D3 mRNA showed that the D3 transcript is expressed in roots, developing leaves, the vegetative meristem, and suspension culture cells. We detected reduced D3 mRNA levels in the mutant allele d3.5.