The protein product of the JIL-1 gene is a tandem chromosomal kinase (referred to as JIL-1). Studies of JIL-1 P-element deletion and insertion loss-of-function mutations suggest that JIL-1 plays a role in histone H3 serine 10 phosphorylation, polytene chromosome morphology maintenance, dosage compensation pathways, and homeotic transformation pathways. Due to the limitation of deletion/insertion mutations, EMS mutagenesis was performed to induce point mutations of JIL-1 and the mutation screen was designed to recover temperature-sensitive JIL-1 alleles. 11 new mutant alleles were identified and 4 were selected for further analysis. The mutant phenotypes given by the four new JIL-1 alleles were characterized. Three of the four showed polytene chromosome phenotypes similar to those of individuals homozygous for a null allele of JIL-1, and one showed polytene chromosome phenotypes similar to those of individuals homozygous for a hypomorphic allele of JIL-1. All of the four mutations produce an adult male homeotic transformation phenotype similar to that of known JIL-1 hypomorphic alleles, even though the penetrance of this phenotype is not the same for each of the four mutations. Fluorescence-conjugated antibody staining of mutant chromosomes showed that for all four mutations the JIL-1 protein level was reduced and the localization of the JIL-1 staining was different from that of the wild type controls. Western blot analysis showed no detectable amounts of JIL-1 protein in mutant individuals for all four mutations. The DNA sequence of the JIL-1 gene of one of the new mutants, 29-1b, was determined and no significant base sequence changes were found in the open reading frame.