This paper describes a rapid and simple method for isolation of medullary thick ascending limbs (MTAL) from rat kidney. The technique takes advantage of the fact that MTAL represents a high fraction of the inner stripe (IS) tissue in the outer medulla, and that this nephron segment is more resistant than others to mechanical and enzymatic disruption. Special attention was given in the design of each step of the isolation procedure in order to improve purity and yield of the preparation. Major steps are the following: careful dissection of the IS; cutting IS tissue into small pieces of regular size (approximately equal to 1 mm3); mild and brief enzymatic hydrolysis in a 65 U/ml collagenase solution; separation of long MTAL segments from other tubule fragments and cells, and washing of the collagenase solution, on a nylon sieve (100 microns opening). This technique does not require lengthy centrifugations and provides about 6 mg fresh tissue (= 1 mg protein) from two rat kidneys in 2 h. Light microscopy and transmission electron microscopy show a good purity (at least 95%) and good preservation of TAL ultrastructural morphology. Adenylate cyclase responsiveness to arginine-vasopressin (AVP), glucagon (GLU) and salmon calcitonin (SCT) of the MTAL suspension is similar to that reported for single microdissected rat MTAL. Viability of the MTALs was demonstrated by the ability to accumulate cyclic AMP in presence of AVP, GLU, SCT and forskolin. Normal oxygen consumption was 45.1 +/- 2.4 (SEM) microliter . mg protein-1 . h-1 (n = 8).(ABSTRACT TRUNCATED AT 250 WORDS)