HL60 cells isolated for resistance to Adriamycin are multidrug resistant and defective in the cellular accumulation of drug. These cells do not however overexpress mdr1 and do not contain detectable levels of P-glycoprotein. In the present study we have prepared antisera against synthetic peptides that correspond to various sequence domains of P-glycoprotein and have examined by Western blot analysis the reactivity of these antisera with proteins contained in membranes of HL60/Adr cells. All antisera are highly reactive with a Mr 180,000 (p180) P-glycoprotein contained in membranes of HL60 cells isolated for resistance to vincristine (HL60/Vinc). In contrast, of 13 antisera tested 12 do not react with any resistance-associated protein in the HL60/Adr isolate. One antiserum (ASP14) is however highly reactive with a Mr 190,000 protein (p190) contained in HL60/Adr membranes. This protein is not detected in drug-sensitive cells. ASP14 also reacts with proteins p195 and p50 contained in a second independent HL60/Adr isolate. Analysis of membrane subfractions shows that p190 is located primarily in the endoplasmic reticulum with only low levels contained in plasma membranes. Additional studies demonstrate that endoplasmic reticulum of HL60/Adr cells contain a major Mr 190,000 protein that is capable of binding the photoaffinity agent 8-azido[alpha-32P]ATP. p195 contained in a second HL60/Adr isolate is also labeled with 8-azido[alpha-32P]ATP. These results thus demonstrate that antiserum against a specific P-glycoprotein sequence detects a p190 (p195) resistance-associated membrane protein in two independent HL60/Adr isolates. p190 (p195) and P-glycoprotein thus contain a minor sequence homology and based on the specificity of ASP14 this occurs in a region which may be involved in nucleotide binding. Possibly this sequence is common to and essential for the functionality of proteins which contribute to resistance by reducing cellular drug levels.