The alpha and beta tubulin genes of Leishmania major were cloned and used to study the genomic organization, chromosomal location and transcription of tubulin genes in L. major. The number of beta tubulin isogenes was determined by hybridization of probes representing the 5' and 3' ends of the cloned beta tubulin cDNA sequence to genomic Southern blots which showed that four complete isogenes exist on Ava1 fragments of size 4.4, 3.9, 1.85 and 1.7 kilobase pairs (kb). These genes are present at a relative ratio of 1:18:3:1 with the 3.9 kb fragment being tandemly repeated. The chromosomal location of each tubulin isogene was studied by purification of individual chromosomes fractionated by pulsed field gradient (PFG) gel electrophoresis. Using an improved PFG procedure, L. major contains at least 23 chromosome-sized bands some of which are present in non-stoichiometric amounts, suggesting that there are more than 23 individual chromosomes. The alpha tubulin genes are located on chromosome 9. The 3.9 kb beta tubulin cluster and the 1.7 kb isogene are linked on chromosome 21 and two dispersed beta tubulin isogenes exist on chromosomes 7 and 13. Thus, three non-allelic beta tubulin loci exist in L. major. Analysis of tubulin transcripts revealed a single abundant alpha tubulin RNA (2050 nucleotides, nt) but three abundant beta tubulin RNAs (2200, 2800, 3400 nt). The 2200 nt RNA is transcribed from the tandemly clustered beta tubulin isogene on chromosome 21. The 2800 nt and 3400 nt RNAs appear to represent additional transcripts from one or both of the dispersed beta tubulin isogenes on chromosomes 13 and 7.